Therapeutic Enzyme Formulations And Uses Thereof

ABSTRACT

Pharmaceutical formulations of glutenase enzymes are provided. The enzymes find particular use in the treatment of a Celiac or dermatitis herpetiformis patient.

This application is related to U.S. Provisional 60/565,668, filed Apr.26, 2004; to U.S. Provisional application 60/357,238 filed Feb. 14,2002; to U.S. Provisional Application 60/380,761 filed May 14, 2002; toU.S. Provisional Application 60/392,782 filed Jun. 28, 2002; and to U.S.Provisional application No. 60/422,933, filed Oct. 31, 2002, to U.S.Provisional Application 60/428,033, filed Nov. 20, 2002, to U.S.Provisional Application 60/435,881, filed Dec. 20, 2002, and to U.S.Ser. No. 10/367,405, filed Feb. 14, 2004, each of which are hereinspecifically incorporated by reference.

BACKGROUND OF THE INVENTION

In 1953, it was first recognized that ingestion of gluten, a commondietary protein present in wheat, barley and rye causes disease insensitive individuals. Gluten is a complex mixture of glutamine- andproline-rich glutenin and prolamine molecules, which is thought to beresponsible for disease induction. Ingestion of such proteins bysensitive individuals produces flattening of the normally luxurious,rug-like, epithelial lining of the small intestine known to beresponsible for efficient and extensive terminal digestion of peptidesand other nutrients. Clinical symptoms of Celiac Sprue include fatigue,chronic diarrhea, malabsorption of nutrients, weight loss, abdominaldistension, anemia, as well as a substantially enhanced risk for thedevelopment of osteoporosis and intestinal malignancies (lymphoma andcarcinoma). The disease has an incidence of approximately 1 in 200 inEuropean populations.

A related disease is dermatitis herpetiformis, which is a chroniceruption characterized by clusters of intensely pruritic vesicles,papules, and urticaria-like lesions. IgA deposits occur in almost allnormal-appearing and perilesional skin. Asymptomatic gluten-sensitiveenteropathy is found in 75 to 90% of patients and in some of theirrelatives. Onset is usually gradual. Itching and burning are severe, andscratching often obscures the primary lesions with eczematization ofnearby skin, leading to an erroneous diagnosis of eczema. Strictadherence to a gluten-free diet for prolonged periods may control thedisease in some patients, obviating or reducing the requirement for drugtherapy. Dapsone, sulfapyridine and colchicines are sometimes prescribedfor relief of itching.

Celiac Sprue is generally considered to be an autoimmune disease and theantibodies found in the serum of the patients supports a theory of animmunological nature of the disease. Antibodies to tissuetransglutaminase (tTG) and gliadin appear in almost 100% of the patientswith active Celiac Sprue, and the presence of such antibodies,particularly of the IgA class, has been used in diagnosis of thedisease.

The large majority of patients express the HLA-DQ2 [DQ(a1*0501, b1*02)]and/or DQ8 [DQ(a1*0301, b1*0302)] molecules. It is believed thatintestinal damage is caused by interactions between specific gliadinoligopeptides and the HLA-DQ2 or DQ8 antigen, which in turn induceproliferation of T lymphocytes in the sub-epithelial layers. T helper 1cells and cytokines apparently play a major role in a local inflammatoryprocess leading to villus atrophy of the small intestine.

At the present time there is no good therapy for the disease, except tocompletely avoid all foods containing gluten. Although gluten withdrawalhas transformed the prognosis for children and substantially improved itfor adults, some people still die of the disease, mainly adults who hadsevere disease at the outset. An important cause of death islymphoreticular disease (especially intestinal lymphoma). It is notknown whether a gluten-free diet diminishes this risk. Apparent clinicalremission is often associated with histologic relapse that is detectedonly by review biopsies or by increased EMA titers.

Gluten is so widely used, for example in commercial soups, sauces, icecreams, hot dogs, and other foods, that patients need detailed lists offoodstuffs to avoid and expert advice from a dietitian familiar withceliac disease. Ingesting even small amounts of gluten may preventremission or induce relapse. Supplementary vitamins, minerals, andhematinics may also be required, depending on deficiency. A few patientsrespond poorly or not at all to gluten withdrawal, either because thediagnosis is incorrect or because the disease is refractory. In thelatter case, oral corticosteroids (e.g., prednisone 10 to 20 mg bid) mayinduce response.

In view of the serious and widespread nature of Celiac Sprue, improvedmethods of treating or ameliorating the effects of the disease areneeded. The present invention addresses such needs.

SUMMARY OF THE INVENTION

The present invention provides glutenase enzymes and enzyme formulationsuseful in the treatment of Celiac Sprue and/or dermatitis herpetiformis.The enzymes decrease the levels of toxic gluten oligopeptides infoodstuffs, either prior to or after ingestion by a patient. Enzymes ofinterest include prolyl endopeptidases (PEP), e.g. the Myxococcusxanthus PEP; and endoprotease, e.g. Hordeum vulgare subsp. vulgare EPB2,biologically active fragments or derivatives thereof. Certain glutenoligopeptides known to be resistant to cleavage by gastric andpancreatic enzymes are digested by such enzymes, thereby preventing orrelieving their toxic effects in Celiac Sprue or dermatitisherpetiformis patients.

In one embodiment, the invention provides purified Myxococcus xanthusPEP, biologically active fragments or derivatives thereof; andpharmaceutical formulations thereof. The enzyme can be expressed in aheterologous host cell, e.g. a heterologous bacteria, and purified byaffinity chromatography. It is found that the enzyme can be purified,lyophilized, and formulated into unit does, such as tablets, entericcoated capsules, etc., while substantially retaining biologicalactivity. Formulations of interest include formulations in which theenzyme is contained within an enteric coating that allows delivery ofthe active agent to the intestine and formulations in which the activeagents are stabilized to resist digestion in acidic stomach conditions.The formulation may comprise one or more enzymes or a mixture or“cocktail” of agents having different activities.

These and other aspects and embodiments of the invention are describedin more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of pH on the turnover numbers (kcat) of FM PEP, MX PEPand SC PEP.

FIG. 2. Resistance of the FM PEP and the MX PEP to inactivation bygastric and pancreatic enzymes. Pancreatic enzyme stability wasevaluated by treating 5 U/ml of the FM PEP and the MX PEP with 1 mg/mltrypsin, 1 mg/ml chymotrypsin, 0.2 mg/ml elastase and 0.2 mg/mlcarboxypeptidase A (40 mM phosphate, pH=6.5). Pepsin stability wastested by treating the FM PEP and the MX PEP (5 U/ml) with 1 mg/mlpepsin (pH=2, 20 mM HCl).

FIG. 3. Site specificity of PQPQLPYPQPQLP hydrolysis by individual PEPs.HPLC-UV (215 nm) traces are shown for each reaction mixture. Initialcleavage fragments (100 μM (SEQ ID NO:11) PQPQLPYPQPQLP, 0.1 μM enzyme,t=5 min) were identified by tandem mass spectrometry. The startingmaterial (SEQ ID NO:11) PQPQLPYPQPQLP and the cleavage fragments A: (SEQID NO:11, aa. 1-8) PQPQLPYP, B: (SEQ ID NO:11, aa 7-13) YPQPQLP, C: (SEQID NO:12, aa 1-6) PQPQLP, D: (SEQ ID NO:11, aa 2-6) QPQLP) are indicatedin the traces.

FIGS. 4A-4C. Hydrolysis of (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF by FM PEP, MX PEP and SC PEP. (A) Time dependence ofhydrolysis in the presence of 10 μM substrate and 0.1 μM enzyme. Thesubstrate appears as a doublet at a retention time of ca. 18 min, due tothe presence of equal quantities of the 32-mer from which the N-terminalLeu is deleted; presence of this contaminant does not affect analysis.From the residual peak areas, the rates of substrate (33-mer+32-mer)disappearance were calculated as 2.3 μM/min (FM PEP), 0.43 μM/min (MXPEP) and 0.07 μM/min (SC PEP). (B) Initial cleavage fragments observeddue to hydrolysis by FM PEP (t=1 min) and MX PEP (t=5 min). (C) Summaryof initial cleavage fragments from FM PEP and MX PEP catalyzedhydrolysis of the 33-mer substrate.

FIGS. 5A-5C. Competitive proteolysis of (SEQ ID NO:11) PQPQLPYPQPQLP and(SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF by each PEP. 10 μM ofthe longer peptide and 50 μM of the shorter peptide were co-incubatedwith 0.1 μM of (A) FM PEP; (B) MX PEP; (C) SC PEP.

FIGS. 6A-6B. Competitive proteolysis of (SEQ ID NO:11) PQPQLPYPQPQLP (50μM) and (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (10 μM) in thepresence of 30 mg/ml pepsin-treated gluten. This complex mixture ofsubstrates was treated under physiological conditions with a mixture ofpancreatic enzymes (trypsin, chymotrypsin, carboxypeptidase, elastase),brush border membrane enzymes (derived from rat small intestine) andeither (A) FM PEP or (B) MX PEP.

FIG. 7. (SEQ ID NO:12) Proteolysis of LQLQPFPQPQLPYPQPQLPYPQPQL PYPQPQPF(5 μM) co-perfused with individual PEP's (0.1 μM) in the smallintestinal lumen of an anesthetized rat. Each enzyme-substrate mixturewas introduced via a catheter into a 15-20 cm segment of the upperjejunum. Samples were collected at the other end of the segment, andanalyzed by UV-HPLC (215 nm). The control without any PEP is shown inthe top trace.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Glutenase enzymes and enzyme formulations are useful in the treatment ofgluten intolerance. The enzymes decrease the levels of toxic glutenoligopeptides in foodstuffs, either prior to or after ingestion by apatient. Enzymes of interest include prolyl endopeptidases (PEP), e.g.the Myxococcus xanthus PEP; and endoproteases, e.g. Hordeum vulgaresubsp. vulgare EPB2. Certain gluten oligopeptides known to be resistantto cleavage by gastric and pancreatic enzymes are digested by suchenzymes, thereby preventing or relieving their toxic effects inpatients. Gluten intolerance is associated primarily with Celiac Sprueand dermatitis herpetiformis, however it is also known in the art to befound in other patients, e.g. associated with autism. Such patients mayalso be treated with the methods of the invention.

In some patients, these methods and compositions allow the patient toingest glutens without serious health consequences, much the same asindividuals that do not suffer from either of these conditions. In someembodiments, the formulations of the invention comprise a glutenasecontained in an enteric coating that allows delivery of the activeagent(s) to the intestine; in other embodiments, the active agent(s) isstabilized to resist digestion in acidic stomach conditions. In somecases the active agent(s) have hydrolytic activity under acidic pHconditions, and can therefore initiate the proteolytic process on toxicgluten sequences in the stomach itself. Alternative methods ofadministration provided by the invention include genetic modification ofpatient cells, e.g. enterocytes, to express increased levels ofglutenases; and the introduction of micro-organisms expressing suchglutenases so as to transiently or permanently colonize the patient'sintestinal tract. Such modified patient cells (which include cells thatare not derived from the patient but that are not immunologicallyrejected when administered to the patient) and microorganisms of theinvention are, in some embodiments, formulated in a pharmaceuticallyacceptable excipient, or introduced in foods. In another embodiment, theinvention provides foods pretreated or combined with a glutenase andmethods for treating foods to remove the toxic oligopeptides of gluten.

The compositions of the invention can be used for prophylactic as wellas therapeutic purposes. As used herein, the term “treating” refers bothto the prevention of disease and the treatment of a disease or apre-existing condition. The invention provides a significant advance inthe treatment of ongoing disease, to stabilize or improve the clinicalsymptoms of the patient. Such treatment is desirably performed prior toloss of function in the affected tissues but can also help to restorelost function or prevent further loss of function. Evidence oftherapeutic effect may be any diminution in the severity of disease,particularly as measured by the severity of symptoms such as fatigue,chronic diarrhea, malabsorption of nutrients, weight loss, abdominaldistension, anemia, and other symptoms of Celiac Sprue. Other diseaseindicia include the presence of antibodies specific for glutens, thepresence of antibodies specific for tissue transglutaminase, thepresence of pro-inflammatory T cells and cytokines, damage to the villusstructure of the small intestine as evidenced by histological or otherexamination, enhanced intestinal permeability, and the like.

Patients that may be treated by the methods of the invention includethose diagnosed with celiac sprue through one or more of serologicaltests, e.g. anti-gliadin antibodies, anti-transglutaminase antibodies,anti-endomysial antibodies; endoscopic evaluation, e.g. to identifyceliac lesions; histological assessment of small intestinal mucosa, e.g.to detect villous atrophy, crypt hyperplasia, infiltration ofintra-epithelial lymphocytes; and any GI symptoms dependent on inclusionof gluten in the diet.

Given the safety of oral proteases, they also find a prophylactic use inhigh-risk populations, such as Type I diabetics, family members ofdiagnosed celiac patients, HLA-DQ2 positive individuals, and/or patientswith gluten-associated symptoms that have not yet undergone formaldiagnosis. Such patients may be treated with regular-dose or low-dose(10-50% of the regular dose) enzyme. Similarly, temporary high-dose useof such an agent is also anticipated for patients recovering fromgluten-mediated enteropathy in whom gut function has not yet returned tonormal, for example as judged by fecal fat excretion assays.

Patients that can benefit from the present invention may be of any ageand include adults and children. Children in particular benefit fromprophylactic treatment, as prevention of early exposure to toxic glutenpeptides can prevent initial development of the disease. Childrensuitable for prophylaxis can be identified by genetic testing forpredisposition, e.g. by HLA typing; by family history, by T cell assay,or by other medical means. As is known in the art, dosages may beadjusted for pediatric use.

The methods of the invention, as well as tests to determine theirefficacy in a particular patient or application, can be carried out inaccordance with the teachings herein using procedures standard in theart. Thus, the practice of the present invention may employ conventionaltechniques of molecular biology (including recombinant techniques),microbiology, cell biology, biochemistry and immunology within the scopeof those of skill in the art. Such techniques are explained fully in theliterature, such as, “Molecular Cloning: A Laboratory Manual”, secondedition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J.Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987);“Methods in Enzymology” (Academic Press, Inc.); “Handbook ofExperimental Immunology” (D. M. Weir & C. C. Blackwell, eds.); “GeneTransfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds.,1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al.,eds., 1987); “PCR: The Polymerase Chain Reaction” (Mullis et al., eds.,1994); and “Current Protocols in Immunology” (J. E. Coligan et al.,eds., 1991); as well as updated or revised editions of all of theforegoing.

As used herein, the term “glutenase” refers to an enzyme useful in themethods of the present invention that is capable, alone or incombination with endogenous or exogenously added enzymes, of cleavingtoxic oligopeptides of gluten proteins of wheat, barley, oats and ryeinto non-toxic fragments. Gluten is the protein fraction in cerealdough, which can be subdivided into glutenins and prolamines, which aresubclassified as gliadins, secalins, hordeins, and avenins from wheat,rye, barley and oat, respectively. For further discussion of glutenproteins, see the review by Wieser (1996) Acta Paediatr Suppl. 412:3-9,incorporated herein by reference.

ENZYMES

In one embodiment of the present invention, the glutenase enzyme is aPEP. Homology-based identification (for example, by a PILEUP sequenceanalysis) of prolyl endopeptidases can be routinely performed by thoseof skill in the art upon contemplation of this disclosure to identifyPEPs suitable for use in the methods of the present invention. PEPs areproduced in microorganisms, plants and animals. PEPs belong to theserine protease superfamily of enzymes and have a conserved catalytictriad composed of a Ser, His, and Asp residues. Some of these homologshave been characterized, e.g. the enzymes from F. meningoscepticum,Aeromonas hydrophila, Aeromonas punctata, Novosphingobium capsulatum,Pyrococcus furiosus and from mammalian sources are biochemicallycharacterized PEPs. Others such as the Nostoc and Arabidopsis enzymesare likely to be PEPs but have not been fully characterized to date.Homologs of the enzymes of interest may be found in publicly availablesequence databases, and the methods of the invention include suchhomologs. Candidate enzymes are expressed using standard heterologousexpression technologies, and their properties are evaluated using theassays described herein.

In one embodiment of the invention, the glutenase is Flavobacteriummeningosepticum PEP (Genbank ID # D10980). Relative to the F.meningoscepticum enzyme, the pairwise sequence identity of this familyof enzymes is in the 30-60% range. Accordingly, PEPs include enzymeshaving >30% identity to the F. meningoscepticum enzyme (as in thePyrococcus enzymes), or having >40% identity (as in the Novosphingobiumenzymes), or having >50% identity (as in the Aeromonas enzymes) to theF. meningoscepticum enzyme. A variety of assays have verified thetherapeutic utility of this PEP. In vitro, this enzyme has been shown torapidly cleave several toxic gluten peptides, including the highlyinflammatory 33-mer, (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.In vivo it acts synergistically with the peptidases of the intestinalbrush border membrane so as to rapidly detoxify these peptides, as wellas gluten that has been pre-treated with gastric and pancreaticproteases. It has broad chain length specificity, making it especiallywell suited for the breakdown of long proline-rich peptides releasedinto the duodenum from the stomach. The enzyme has a pH optimum aroundpH 7, and has high specific activity under conditions that mimic theweakly acidic environment of the upper small intestine. FlavobacteriumPEP can cleave all T cell epitopes in gluten that have been tested todate. It has particular preference for the immunodominant epitopes foundin alpha-gliadin. When grocery-store gluten is treated with this PEP, arapid decrease in its antigenicity can be observed, as judged by LC-MSanalysis and testing against polyclonal T cell lines derived from smallintestinal biopsies from Celiac Sprue patients. The denatured protein isnon-allergenic in rodents, rabbits and humans. It is relatively stabletoward destruction by pancreatic proteases, an important feature sinceunder physiological conditions it will be expected to act in concertwith those enzymes.

Another enzyme of interest is Sphingomonas capsulata PEP (Genbank ID#AB010298). This enzyme is comparable to the Flavobacterium andMyxococcus enzyme. It has broader sequence specificity than either theFlavobacterium or the Myxococcus PEP, and may therefore be able todestroy the widest range of antigenic epitopes. Like the Myxococcusenzyme, it is also well expressed in E. coli.

Another enzyme of interest is Penicillium citrinum PEP (Genbank ID#D25535). This enzyme has been shown to possess PEP activity based on itsability to effectively cleave a number of Pro-Xaa bonds in peptides suchas dynorphin A and substance P. The putative metalloprotease has theadvantages of small size and a pH profile that renders it suitable toworking in concert with the pancreatic enzymes in the duodenum. As such,it is a good candidate for the treatment of Celiac Sprue.

Another enzyme of interest is Lactobacillus helveticus PEP (Genbank ID#321529). Unlike the above PEPs, this PEP is a zinc enzyme. It canefficiently proteolyze long peptide substrates such as the caseinpeptides YQEPVLGPVRGPFPIIV and RPKHPIKHQ. Proteolysis occurs at all PVand PI subsites, suggesting the PEP prefers hydrophobic residues at theS1′ position, as are frequently found in gluten. Since the producerstrain of L. helveticus CNRZ32 is commonly used in cheesemaking, thisenzyme has desirable properties as a food-grade enzyme.

Another enzyme of interest is Myxococcus xanthus PEP (Genbank ID#AF127082). This enzyme possesses many of the advantages of theFlavobacterium PEP. It can cleave the 33-mer into small non-toxicpeptides. Whereas the Flavobacterium enzyme appears to have a relativelystrict preference for PQ bonds in gliadin peptides, the Myxococcusenzyme can cleave at PQ, PY and PF bonds, a feature that allows it toproteolyze a broader range of gluten epitopes. Compared to theFlavobacterium enzyme, it has equivalent stability toward the pancreaticproteases and superior stability toward acidic environments. TheMyxococcus enzyme is well expressed in E. coli, making it feasible toproduce this enzyme cheaply.

Glutenase enzyme fragments of interest include fragments of at leastabout 20 contiguous amino acids, more usually at least about 50contiguous amino acids, and may comprise 100 or more amino acids, up tothe complete protein, and may extend further to comprise additionalsequences. In each case, the key criterion is whether the fragmentretains the ability to digest the toxic oligopeptides that contribute tothe symptoms of Celiac Sprue.

Modifications of interest that do not alter primary sequence includechemical derivatization of proteins, including, for example, acylation,e.g. lauryl, stearyl, myrsityl, decyl, etc. groups, PEGylation,esterification, or amidation. Such modifications may be used to increasethe resistance of the enzyme toward proteolysis, e.g. by attachment ofPEG sidechains or lauryl groups to surface lysines. Also included aremodifications of glycosylation, e.g. those made by modifying theglycosylation patterns of a protein during its synthesis and processingor in further processing steps; e.g. by exposing the protein to enzymesthat affect glycosylation, such as mammalian glycosylating ordeglycosylating enzymes. Also embraced are sequences that havephosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine,or phosphothreonine.

The amino acid sequence of a glutenase, e.g. a naturally occurringglutenase, can be altered in various ways known in the art to generatetargeted changes in sequence and additional glutenase enzymes useful inthe formulations and compositions of the invention. Such variants willtypically be functionally-preserved variants, which differ, usually insequence, from the corresponding native or parent protein but stillretain the desired biological activity. Variants also include fragmentsof a glutenase that retain enzymatic activity. Various methods known inthe art can be used to generate targeted changes, e.g. phage display incombination with random and targeted mutations, introduction of scanningmutations, and the like.

A variant can be substantially similar to a native sequence, i.e.differing by at least one amino acid, and can differ by at least two butusually not more than about ten amino acids (the number of differencesdepending on the size of the native sequence). The sequence changes maybe substitutions, insertions or deletions. Scanning mutations thatsystematically introduce alanine, or other residues, may be used todetermine key amino acids. Conservative amino acid substitutionstypically include substitutions within the following groups: (glycine,alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid);(asparagine, glutamine); (serine, threonine); (lysine, arginine); and(phenylalanine, tyrosine).

Various modifications may be made to the enzyme sequence. The MX PEP hasa similar structure as that from the porcine muscle or brain. The enzymeconsists of a catalytic domain with a typical α/β hydrolase fold, whichis covalently connected to a cylindrical barrel-shaped propeller domain.The catalytic domain is made up of N-terminal residues 1-67 andC-terminal residues 410-678; the propeller domain includes residues71-406. Two linear strands formed by residues 67-70 and 407-409covalently connect the two domains. This analysis of the M. xanthus PEPis useful in the design of modified enzymes. Typically such modifiedenzymes will retain the catalytic triad (Ser 533, Asp 616 and His 651)as well as the conserved Arg 618 residues, all of which are expected tobe important for activity. Residues Asn534, Tyr453 and Arg618 areconserved in the prolyl endopeptidase family, and such residues may alsobe conserved in the design of modified enzymes. In one embodiment, theextended region of the propeller domain is replaced with a shortflexible linker, e.g. comprised of 5-10 Gly residues, thereby truncatingthe protein and reducing its proteolytic susceptibility to pepsin.

For example, mutations have been made with V458 and G532, which areclose to the catalytic Ser533 in the binding pocket. Their mutantsretain wild-type activity toward Suc-Ala-Pro-pNA, but show reducedactivity toward the 13-mer. Other mutants, R572A/Q, I575A and F229Y havean increased specificity for a longer substrate.

Enzymes modified to provide for a specific characteristic of interestmay be further modified, for e.g. by mutagenesis, exon shuffling, etc.,as known in the art, followed by screening or selection, so as tooptimize or restore the activity of the enzyme, e.g. to wild-typelevels.

Also useful in the practice of the present invention are proteins thathave been modified using molecular biological techniques and/orchemistry so as to improve their resistance to proteolytic degradationand/or to acidic conditions such as those found in the stomach, and tooptimize solubility properties or to render them more suitable as atherapeutic agent. For example, the backbone of the peptidase can becyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem.275:23783-23789). Analogs of such proteins include those containingresidues other than naturally occurring L-amino acids, e.g. D-aminoacids or non-naturally occurring synthetic amino acids.

The glutenase proteins useful in the practice of the present inventionmay also be isolated and purified in accordance with conventionalmethods from recombinant production systems and from natural sources.Protease production can be achieved using established host-vectorsystems in organisms such as E. coli, S. cerevisiae, P. pastoris,Lactobacilii, Bacilli and Aspergilli. Integrative or self-replicativevectors may be used for this purpose. In some of these hosts, theprotease is expressed as an intracellular protein and subsequentlypurified, whereas in other hosts the enzyme is secreted into theextracellular medium. Purification of the protein can be performed by acombination of ion exchange chromatography, Ni-affinity chromatography(or some alternative chromatographic procedure), hydrophobic interactionchromatography, and/or other purification techniques. Typically, thecompositions used in the practice of the invention will comprise atleast 20% by weight of the desired product, more usually at least about50% by weight, preferably at least about 85% by weight, at least about90%, and for therapeutic purposes, may be at least about 95% by weight,in relation to contaminants related to the method of preparation of theproduct and its purification. Usually, the percentages will be basedupon total protein. Proteins in such compositions may be present at aconcentration of at least about 500 μg/ml; at least about 1 mg/mg; atleast about 5 mg/ml; at least about 10 mg/ml, or more.

In one aspect, the present invention provides a purified preparation ofa glutenase. Such enzymes may be produced by recombinant methods. In oneembodiment, such methods utilize a bacterial host for expression,although fungal and eukaryotic systems find use for some purposes.Coding sequences that contain a signal sequence, or that are engineeredto contain a signal sequence can be secreted into the periplasmic spaceof a bacterial host. An osmotic shock protocol can then be used torelease the periplasmic proteins into the supernatant.

Where the enzyme is a cytoplasmic enzyme, a signal sequence can beintroduced for periplasmic secretion, or the enzyme can be isolated froma cytoplasmic lysate. Methods for purification include Ni-NTA affinitypurification, e.g. in combination with introduction of a histidine tag;and chromatography methods known in the art, e.g. cation exchange, anionexchange, gel filtration, HPLC, FPLC, and the like.

For various purposes, such as stable storage, the enzyme may belyophilized. Lyophilization is preferably performed on an initiallyconcentrated preparation, e.g. of at least about 1 mg/ml. Peg may beadded to improve the enzyme stability. It has been found that MX PEP canbe lyophilized without loss of specific activity. The lyophilized enzymeand excipients is useful in the production of enteric-coated capsules ortablets, e.g. a single capsule or tablet may contain at least about 1mg. PEP, usually at least about 10 mg PEP, and may contain at least 100mg PEP, at least about 500 mg PEP, or more. As described in detail here,enteric coatings may be applied, where a substantial fraction of theactivity is retained, and is stable for at least about 1 month at 4° C.It has also been found that MX PEP retains activity in a tabletformulation.

Prior to the present invention, there was no perceived need for aglutenase that could be ingested by a human or mixed with a foodstuff.Thus, prior to the present invention most glutenases did not exist in aform free of contaminants that could be deleterious to a human ifingested. The present invention creates a need for such glutenasepreparations and provides them and methods for preparing them. In arelated embodiment, the present invention provides novel foodstuffs thatare derived from gluten-containing foodstuffs but have been treated toreduce the concentration and amount of the oligopeptides andoligopeptide sequences discovered to be toxic to Celiac Sprue patients.While gluten-free or reduced-gluten content foods have been made, thefoodstuffs of the present invention differ from such foodstuffs not onlyby the manner in which they are prepared, by treatment of the foodstuffwith a glutenase, but also by their content, as the methods of the priorart result in alteration of non-toxic (to Celiac Sprue patients)components of the foodstuff, resulting in a different taste andcomposition. Prior art foodstuffs include, for example, CodexAlimentarius wheat starch, which is available in Europe and has <100 ppmgluten. The starch is usually prepared by processes that take advantageof the fact that gluten is insoluble in water whereas starch is soluble.

In one embodiment, the term “glutenase” as used herein refers to aprotease or a peptidase enzyme that meets one or more of the criteriaprovided herein. Such criteria also find use in the evaluation of enzymemodifications, e.g. as a screening tool following generation ofmodification. In some embodiments, modifications to the MX PEP aminoacid sequence or non-peptidic modifications are assessed using suchassays. Using these criteria, one of skill in the art can determine thesuitability of a candidate enzyme or enzyme modification for use in themethods of the invention. Many enzymes will meet multiple criteria,including two, three, four or more of the criteria, and some enzymeswill meet all of the criteria. The terms “protease” or “peptidase” canrefer to a glutenase and as used herein describe a protein or fragmentthereof with the capability of cleaving peptide bonds, where thescissile peptide bond may either be terminal or internal inoligopeptides or larger proteins. Prolyl-specific peptidases areglutenases useful in the practice of the present invention.

Glutenases of the invention include protease and peptidase enzymeshaving at least about 20% sequence identity at the amino acid level,more usually at least about 40% sequence identity, and preferably atleast about 70% sequence identity to one of the following peptidases:prolyl endopeptidase (PEP) from F. meningosepticum (Genbank accessionnumber D10980), PEP from A. hydrophila (Genbank accession numberD14005), PEP form S. capsulata (Genbank accession number AB010298), DCPI from rabbit (Genbank accession number X62551), DPP IV from Aspergillusfumigatus (Genbank accession number U87950), carboxypeptidase fromAspergillus saitoi (GenBank ID# D25288), PEP from Lactobacillushelveticus (Genbank ID# 321529) or cysteine proteinase B from Hordeumvulgare (Genbank accession number JQ1110).

Each of the above proteases described herein can be engineered toimprove desired properties such as enhanced specificity toward toxicgliadin sequences, improved tolerance for longer substrates, acidstability, pepsin resistance, resistance to proteolysis by thepancreatic enzymes and improved shelf-life. The desired property can beengineered via standard protein engineering methods.

Other than proline, glutamine residues are also highly prevalent ingluten proteins. The toxicity of gluten in Celiac Sprue has beendirectly correlated to the presence of specific Gln residues. Therefore,glutamine-specific proteases are also beneficial for the treatment ofCeliac Sprue. Since oats contain proteins that are rich in glutamine butnot especially rich in proline residues, an additional benefit of aglutamine-specific protease is the improvement of oat tolerance in thoseceliac patients who show mild oat-intolerance. An example of such aprotease is the above-mentioned cysteine endoproteinase from gluten.This enzyme cleaves gluten proteins rapidly with a distinct preferencefor post-Gln cleavage. Also of interest is Hordeum vulgare endoprotease(Genbank accession U19384), which has been shown to efficiently digestα2-gliadin. The enzyme is active under acidic conditions, and is usefulas an orally administered dietary supplement. A gluten-containing dietmay be supplemented with orally administered proEPB2, resulting ineffective degradation of immunogenic gluten peptides in the acidicstomach, before these peptides enter the intestine and are presented tothe immune system. Proteins with high sequence similarity to this enzymeare also of interest. An advantage of these enzymes is that they areconsidered as safe for human oral consumption, due to their presence indietary gluten from barley.

Intestinal dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I arethe rate-limiting enzymes in the breakdown of toxic gliadin peptidesfrom gluten. These enzymes are bottlenecks in gluten digestion in themammalian small intestine because (i) their specific activity isrelatively low compared to other amino- and carboxy-peptidases in theintestinal brush border; and (ii) due to their strong sensitivity tosubstrate chain length, they cleave long immunotoxic peptides such asthe 33-mer extremely slowly. Both these problems can be amelioratedthrough the administration of proline-specific amino- andcarboxy-peptidases from other sources. For example the X-Pro dipeptidasefrom Aspergillus oryzae (GenBank ID# BD191984) and the carboxypeptidasefrom Aspergillus saitoi (GenBank ID# D25288) can improve glutendigestion in the Celiac intestine.

A glutenase of the invention includes a peptidase or protease that has aspecific activity of at least 2.5 U/mg, preferably 25 U/mg and morepreferably 250 U/mg for cleavage of a peptide comprising one of more ofthe following motifs: Gly-Pro-pNA, Z-Gly-Pro-pNA (where Z is abenzyloxycarbonyl group), and Hip-His-Leu, where “Hip” is hippuric acid,pNA is para-nitroanilide, and 1 U is the amount of enzyme required tocatalyze the turnover of 1 μmole of substrate per minute. Chromogenicsubstrates may be utilized in screening, e.g. substrates such asCbz-Gly-Pro-pNA or Suc-Ala-Pro-pNA enables identification ofproline-specific proteases. Similar substrates can also be used toidentify glutamine-specific proteases. These assays can be monitored byUV-Vis spectrophotometric methods.

A glutenase of the invention includes an enzyme belonging to any of thefollowing enzyme classifications: EC 3.4.21.26, EC 3.4.14.5, or EC3.4.15.1.

A glutenase of the invention includes an enzyme having a kcat/Km of atleast about 2.5 s⁻¹ M⁻¹, usually at least about 250 s⁻¹ M⁻¹ andpreferably at least about 25000 s⁻¹ for cleavage of any of the followingpeptides, including known T cell epitopes in gluten, under optimalconditions: (SEQ ID NO:1) QLQPFPQPQLPY or PFPQPQLPY, (SEQ ID NO:3)PQPQLPYPQPQLPY or PQPQLPYPQ, (SEQ ID NO:13) QPQQSFPQQQ or PQQSFPQQQ,(SEQ ID NO:14) QLQPFPQPELPY, (SEQ ID NO:15) PQPELPYPQPELPY, (SEQ IDNO:16) QPQQSFPEQQ; (SEQ ID NO: 30) IQPQQPAQL; (SEQ ID NO:31) QQPQQPYPQ;(SEQ ID NO:32) SQPQQQFPQ; (SEQ ID NO:33) QQPFPQQPQ; or (SEQ ID NO:34)PFSQQQQPV. Cleavage of longer, physiologically generated peptidescontaining one or more of the above epitopes may also be assessed, forexample cleavage of the 33-mer from alpha-gliadin, (SEQ ID NO:12)LQLQPF(PQPQLPY)₃PQPQPF, and the 26-mer from gamma-gliadin, (SEQ IDNO:35) FLQPQQPFPQQPQQPYPQQPQQPFPQ. A glutenase of the invention includespeptidase or protease having a specificity kcat/Km>2 mM⁻¹s⁻¹ for thequenched fluorogenic substrate (SEQ ID NO:36) Abz-QPQQP-Tyr(NO₂)-D.These assays can be monitored by HPLC or fluorescence spectroscopy. Forthe latter assays, suitable fluorophores can be attached to the amino-and carboxy-termini of the peptides.

A glutenase useful in the practice of the present invention can beidentified by its ability to cleave a pretreated substrate to removetoxic gluten oligopeptides, where a “pretreated substrate” is a gliadin,hordein, secalin or avenin protein that has been treated withphysiological quantities of gastric and pancreatic proteases, includingpepsin (1:100 mass ratio), trypsin (1:100), chymotrypsin (1:100),elastase (1:500), and carboxypeptidases A and B (1:100). Pepsindigestion may be performed at pH 2 for 20 min., to mimic gastricdigestion, followed by further treatment of the reaction mixture withtrypsin, chymotrypsin, elastase and carboxypeptidase at pH 7 for 1 hour,to mimic duodenal digestion by secreted pancreatic enzymes. Thepretreated substrate comprises oligopeptides resistant to digestion,e.g. under physiological conditions. A glutenase may catalyze cleavageof pepsin-trypsin-chymotrypsin-elastase-carboxypeptidase (PTCEC) treatedgluten such that less than 10% of the products are longer than (SEQ IDNO:3, aa 1-9) PQPQLPYPQ (as judged by longer retention times on a C18reverse phase HPLC column monitored at A₂₁₅).

The ability of a peptidase or protease to cleave a pretreated substratecan be determined by measuring the ability of an enzyme to increase theconcentration of free NH₂-termini in a reaction mixture containing 1mg/ml pretreated substrate and 10 μg/ml of the peptidase or protease,incubated at 37° C. for 1 hour. A glutenase useful in the practice ofthe present invention will increase the concentration of the free aminotermini under such conditions, usually by at least about 25%, moreusually by at least about 50%, and preferably by at least about 100%. Aglutenase includes an enzyme capable of reducing the residual molarconcentration of oligopeptides greater than about 1000 Da in a 1 mg/ml“pretreated substrate” after a 1 hour incubation with 10 μg/ml of theenzyme by at least about 2-fold, usually by at least about 5-fold, andpreferably by at least about 10-fold. The concentration of sucholigopeptides can be estimated by methods known in the art, for examplesize exclusion chromatography and the like.

A glutenase of the invention includes an enzyme capable ofdetoxification of whole gluten, as monitored by polyclonal T cell linesderived from intestinal biopsies of celiac patients; detoxification ofwhole gluten as monitored by LC-MS-MS; and/or detoxification of wholegluten as monitored by ELISA assays using monoclonal antibodies capableof recognizing sequences specific to gliadin.

For example, a glutenase may reduce the potency by which a “pretreatedsubstrate” can antagonize binding of (SEQ ID NO:17) PQPELPYPQPQLP toHLA-DQ2. The ability of a substrate to bind to HLA-DQ is indicative ofits toxicity; fragments smaller than about 8 amino acids are generallynot stably bound to Class II MHC. Treatment with a glutenase thatdigests toxic oligopeptides, by reducing the concentration of the toxicoligopeptides, prevents a mixture containing them from competing with atest peptide for MHC binding. To test whether a candidate glutenase canbe used for purposes of the present invention, a 1 mg/ml solution of“pretreated substrate” may be first incubated with 10 μg/ml of thecandidate glutenase, and the ability of the resulting solution todisplace radioactive (SEQ ID NO:18) PQPELPYPQPQPLP pre-bound to HLA-DQ2molecules can then be quantified, with a reduction of displacement,relative to a non-treated control, indicative of utility in the methodsof the present invention.

A glutenase of the invention includes an enzyme that reduces theanti-tTG antibody response to a “gluten challenge diet” in a CeliacSprue patient by at least about 2-fold, more usually by at least about5-fold, and preferably by at least about 10-fold. A “gluten challengediet” is defined as the intake of 100 g bread per day for 3 days by anadult Celiac Sprue patient previously on a gluten-free diet. Theanti-tTG antibody response can be measured in peripheral blood usingstandard clinical diagnostic procedures, as known in the art.

Excluded from the term “glutenase” are the following peptidases: humanpepsin, human trypsin, human chymotrypsin, human elastase, papayapapain, and pineapple bromelain, and usually excluded are enzymes havinggreater than 98% sequence identity at the amino acid level to suchpeptidases, more usually excluded are enzymes having greater than 90%sequence identity at the amino acid level to such peptidases, andpreferably excluded are enzymes having greater than 70% sequenceidentity at the amino acid level to such peptidases.

Among gluten proteins with potential harmful effect to Celiac Spruepatients are included the storage proteins of wheat, species of whichinclude Triticum aestivum; Triticum aethiopicum; Triticum baeoticum;Triticum militinae; Triticum monococcum; Triticum sinskajae; Triticumtimopheevii; Triticum turgidum; Triticum urartu, Triticum vavilovii;Triticum zhukovskyi; etc. A review of the genes encoding wheat storageproteins may be found in Colot (1990) Genet Eng (N Y) 12:225-41. Gliadinis the alcohol-soluble protein fraction of wheat gluten. Gliadins aretypically rich in glutamine and proline, particularly in the N-terminalpart. For example, the first 100 amino acids of α- and γ-gliadinscontain ˜35% and ˜20% of glutamine and proline residues, respectively.Many wheat gliadins have been characterized, and as there are manystrains of wheat and other cereals, it is anticipated that many moresequences will be identified using routine methods of molecular biology.In one aspect of the present invention, genetically modified plants areprovided that differ from their naturally occurring counterparts byhaving gliadin proteins that contain a reduced content of glutamine andproline residues.

Examples of gliadin sequences include but are not limited to wheat alphagliadin sequences, for example as provided in Genbank, accession numbersAJ133612; AJ133611; AJ133610; AJ133609; AJ133608; AJ133607; AJ133606;AJ133605; AJ133604; AJ133603; AJ133602; D84341.1; U51307; U51306;U51304; U51303; U50984; and U08287. A sequence of wheat omega gliadin isset forth in Genbank accession number AF280605.

For the purposes of the present invention, toxic gliadin oligopeptidesare peptides derived during normal human digestion of gliadins andrelated storage proteins as described above, from dietary cereals, e.g.wheat, rye, barley, and the like. Such oligopeptides are believed to actas antigens for T cells in Celiac Sprue. For binding to Class II MHCproteins, immunogenic peptides are usually from about 8 to 20 aminoacids in length, more usually from about 10 to 18 amino acids. Suchpeptides may include PXP motifs, such as the motif PQPQLP (SEQ ID NO:8).Determination of whether an oligopeptide is immunogenic for a particularpatient is readily determined by standard T cell activation and otherassays known to those of skill in the art.

As demonstrated herein, during digestion, peptidase resistantoligopeptides remain after exposure of glutens, e.g. gliadin, to normaldigestive enzymes. Examples of peptidase resistant oligopeptides areprovided, for example, as set forth in SEQ ID NO:5, 6, 7 and 10. Otherexamples of immunogenic gliadin oligopeptides are described in Wieser(1995) Baillieres Clin Gastroenterol 9(2):191-207, incorporated hereinby reference.

Determination of whether a candidate enzyme will digest a toxic glutenoligopeptide, as discussed above, can be empirically determined. Forexample, a candidate may be combined with an oligopeptide comprising oneor more Gly-Pro-pNA, Z-Gly-Pro-pNA, Hip-His-Leu, Abz-QLP-Tyr(NO₂-PQ,Abz-PYPQPQ-Tyr(NO₂), PQP-Lys(Abz)-LP-Tyr(NO₂)-PQPQLP,PQPQLP-Tyr(NO₂)-PQP-Lys(Abz)-LP motifs; with one or more of theoligopeptides (SEQ ID NO:1) QLQPFPQPQLPY, (SEQ ID NO:3) PQPQLPYPQPQLPY,(SEQ ID NO:13) QPQQSFPQQQ, (SEQ ID NO:14) QLQPFPQPELPY, (SEQ ID NO:15)PQPELPYPQPELPY, (SEQ ID NO:16) QPQQSFPEQQ or (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; or with a pretreated substratecomprising one or more of gliadin, hordein, secalin or avenin proteinsthat have been treated with physiological quantities of gastric andpancreatic proteases. In each instance, the candidate is determined tobe a glutenase of the invention if it is capable of cleaving theoligopeptide. Glutenases that have a low toxicity for human cells andare active in the physiologic conditions present in the intestinal brushborder are preferred for use in some applications of the invention, andtherefore it may be useful to screen for such properties in candidateglutenases.

The oligopeptide or protein substrates for such assays may be preparedin accordance with conventional techniques, such as synthesis,recombinant techniques, isolation from natural sources, or the like. Forexample, solid-phase peptide synthesis involves the successive additionof amino acids to create a linear peptide chain (see Merrifield (1963)J. Am. Chem. Soc. 85:2149-2154). Recombinant DNA technology can also beused to produce the peptide.

The level of digestion of the toxic oligopeptide can be compared to abaseline value. The disappearance of the starting material and/or thepresence of digestion products can be monitored by conventional methods.For example, a detectable marker can be conjugated to a peptide, and thechange in molecular weight associated with the marker is thendetermined, e.g. acid precipitation, molecular weight exclusion, and thelike. The baseline value can be a value for a control sample or astatistical value that is representative a control population. Variouscontrols can be conducted to ensure that an observed activity isauthentic, including running parallel reactions, positive and negativecontrols, dose response, and the like.

Active glutenases identified by the screening methods described hereincan serve as lead compounds for the synthesis of analog compounds toidentify glutenases with improved properties. Identification of analogcompounds can be performed through use of techniques such asself-consistent field (SCF) analysis, configuration interaction (CI)analysis, and normal mode dynamics analysis.

A desirable property in glutenases is stability against gastric (low pHand pepsin) conditions. Glutenases with enhanced gastric stability canbe identified by mutagenesis, followed colony transfer to liquid culturein high-throughput screening formats such as 96-well plates. Followinggrowth, a fraction of the cell culture can be lysed and the lysateincubated for variable durations under simulated gastric conditions(pepsin, pH 2). Thereafter, the lysate can be assayed with a suitablechromogenic substrate (e.g. Cbz-Succinyl-Ala-Pro-pNA). An intense yellowcolor will develop in the presence of extracts with enhanced gastricstability.

FORMULATIONS

In one embodiment of the present invention, a Celiac Sprue patient is,in addition to being provided a glutenase or food treated in accordancewith the present methods, provided an inhibitor of tissuetransglutaminase, an anti-inflammatory agent, an anti-ulcer agent, amast cell-stabilizing agents, and/or and an-allergy agent. Examples ofsuch agents include HMG-CoA reductase inhibitors with anti-inflammatoryproperties such as compactin, lovastatin, simvastatin, pravastatin andatorvastatin; anti-allergic histamine H1 receptor antagonists such asacrivastine, cetirizine, desloratadine, ebastine, fexofenadine,levocetirizine, loratadine and mizolastine; leukotriene receptorantagonists such as montelukast and zafirlukast; COX2 inhibitors such ascelecoxib and rofecoxib; p38 MAP kinase inhibitors such as BIRB-796; andmast cell stabilizing agents such as sodium chromoglycate (chromolyn),pemirolast, proxicromil, repirinast, doxantrazole, amlexanox nedocromiland probicromil.

As used herein, compounds which are “commercially available” may beobtained from commercial sources including but not limited to AcrosOrganics (Pittsburgh Pa.), Aldrich Chemical (Milwaukee Wis., includingSigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), AvocadoResearch (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet(Cornwall, U.K.), Chemservice Inc. (West Chester Pa.), Crescent ChemicalCo. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman Kodak Company(Rochester N.Y.), Fisher Scientific Co. (Pittsburgh Pa.), FisonsChemicals (Leicestershire UK), Frontier Scientific (Logan Utah), ICNBiomedicals, Inc. (Costa Mesa Calif.), Key Organics (Cornwall U.K.),Lancaster Synthesis (Windham N. H.), Maybridge Chemical Co. Ltd.(Cornwall U.K.), Parish Chemical Co. (Orem Utah), Pfaltz & Bauer, Inc.(Waterbury Conn.), Polyorganix (Houston Tex.), Pierce Chemical Co.(Rockford Ill.), Riedel de Haen A G (Hannover, Germany), SpectrumQuality Product, Inc. (New Brunswick, N.J.), TCI America (PortlandOreg.), Trans World Chemicals, Inc. (Rockville Md.), Wako Chemicals USA,Inc. (Richmond Va.), Novabiochem and Argonaut Technology.

Compounds useful for co-administration with the glutenases and treatedfoodstuffs of the invention can also be made by methods known to one ofordinary skill in the art. As used herein, “methods known to one ofordinary skill in the art” may be identified though various referencebooks and databases. Suitable reference books and treatises that detailthe synthesis of reactants useful in the preparation of compounds of thepresent invention, or provide references to articles that describe thepreparation, include for example, “Synthetic Organic Chemistry”, JohnWiley & Sons, Inc., New York; S. R. Sandier et al., “Organic FunctionalGroup Preparations,” 2nd Ed., Academic Press, New York, 1983; H. O.House, “Modern Synthetic Reactions”, 2nd Ed., W. A. Benjamin, Inc. MenloPark, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed.,John Wiley & Sons, New York, 1992; J. March, “Advanced OrganicChemistry: Reactions, Mechanisms and Structure”, 4th Ed.,Wiley-Interscience, New York, 1992. Specific and analogous reactants mayalso be identified through the indices of known chemicals prepared bythe Chemical Abstract Service of the American Chemical Society, whichare available in most public and university libraries, as well asthrough on-line databases (the American Chemical Society, Washington,D.C., www.acs.org may be contacted for more details). Chemicals that areknown but not commercially available in catalogs may be prepared bycustom chemical synthesis houses, where many of the standard chemicalsupply houses (e.g., those listed above) provide custom synthesisservices.

The glutenase proteins of the invention and/or the compoundsadministered therewith are incorporated into a variety of formulationsfor therapeutic administration. In one aspect, the agents are formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and are formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.As such, administration of the glutenase and/or other compounds can beachieved in various ways, usually by oral administration. The glutenaseand/or other compounds may be systemic after administration or may belocalized by virtue of the formulation, or by the use of an implant thatacts to retain the active dose at the site of implantation.

In pharmaceutical dosage forms, the glutenase and/or other compounds maybe administered in the form of their pharmaceutically acceptable salts,or they may also be used alone or in appropriate association, as well asin combination with other pharmaceutically active compounds. The agentsmay be combined, as previously described, to provide a cocktail ofactivities. The following methods and excipients are exemplary and arenot to be construed as limiting the invention.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

In one embodiment of the invention, the oral formulations compriseenteric coatings, so that the active agent is delivered to theintestinal tract. A number of methods are available in the art for theefficient delivery of enterically coated proteins into the smallintestinal lumen. Most methods rely upon protein release as a result ofthe sudden rise of pH when food is released from the stomach into theduodenum, or upon the action of pancreatic proteases that are secretedinto the duodenum when food enters the small intestine. For intestinaldelivery of a PEP and/or a glutamine specific protease, the enzyme isusually lyophilized in the presence of appropriate buffers (e.g.phosphate, histidine, imidazole) and excipients (e.g. cryoprotectantssuch as sucrose, lactose, trehalose). Lyophilized enzyme cakes areblended with excipients, then filled into capsules, which areenterically coated with a polymeric coating that protects the proteinfrom the acidic environment of the stomach, as well as from the actionof pepsin in the stomach. Alternatively, protein microparticles can alsobe coated with a protective layer. Exemplary films are cellulose acetatephthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate,methacrylate copolymers, and cellulose acetate phthalate.

Other enteric formulations comprise engineered polymer microspheres madeof biologically erodable polymers, which display strong adhesiveinteractions with gastrointestinal mucus and cellular linings and cantraverse both the mucosal absorptive epithelium and thefollicle-associated epithelium covering the lymphoid tissue of Peyer'spatches. The polymers maintain contact with intestinal epithelium forextended periods of time and actually penetrate it, through and betweencells. See, for example, Mathiowitz et al. (1997) Nature 386 (6623):410-414. Drug delivery systems can also utilize a core of superporoushydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh etal. (2001) J Control Release 71 (3):307-18.

Gluten detoxification for a gluten sensitive individual can commence assoon as food enters the stomach, since the acidic environment (˜pH 2) ofthe stomach favors gluten solubilization. Introduction of an acid-stablePEP or glutamine-specific protease into the stomach will synergize withthe action of pepsin, leading to accelerated destruction of toxicpeptides upon entry of gluten in the small intestines of celiacpatients. In contrast to a PEP that acts in the small intestine, gastricenzymes need not be formulated with enteric coatings. Indeed, sinceseveral proteases (including the above-mentioned cysteine proteinasefrom barley) self-activate by cleaving the corresponding pro-proteinsunder acidic conditions. In one embodiment of the invention, theformulation comprises a pro-enzyme that is activated in the stomach.

In another embodiment, a microorganism, for example bacterial or yeastculture, capable of producing glutenase is administered to a patient.Such a culture may be formulated as an enteric capsule; for example, seeU.S. Pat. No. 6,008,027, incorporated herein by reference.Alternatively, microorganisms stable to stomach acidity can beadministered in a capsule, or admixed with food preparations.

In another embodiment, the glutenase is admixed with food, or used topre-treat foodstuffs containing glutens. Glutenase present in foods canbe enzymatically active prior to or during ingestion, and may beencapsulated or otherwise treated to control the timing of activity.Alternatively, the glutenase may be encapsulated to achieve a timedrelease after ingestion, e.g. in the intestinal tract.

Formulations are typically provided in a unit dosage form, where theterm “unit dosage form,” refers to physically discrete units suitable asunitary dosages for human subjects, each unit containing a predeterminedquantity of glutenase in an amount calculated sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms of the present invention depend on the particular complex employedand the effect to be achieved, and the pharmacodynamics associated witheach complex in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are commercially available. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are commercially available. Any compound useful inthe methods and compositions of the invention can be provided as apharmaceutically acceptable base addition salt. “Pharmaceuticallyacceptable base addition salt” refers to those salts which retain thebiological effectiveness and properties of the free acids, which are notbiologically or otherwise undesirable. These salts are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, thesodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Preferred inorganicsalts are the ammonium, sodium, potassium, calcium, and magnesium salts.Salts derived from organic bases include, but are not limited to, saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like.Particularly preferred organic bases are isopropylamine, diethylamine,ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Depending on the patient and condition being treated and on theadministration route, the glutenase may be administered in dosages of0.01 mg to 500 mg/kg body weight per day, e.g. about 20 mg/day for anaverage person. Efficient proteolysis of gluten in vivo for an adult mayrequire at least about 500 units of a therapeutically efficaciousenzyme, usually at least about 1000 units, more usually at least about2000 units, and not more than about 50,000 units, usually not more thanabout 20,000 units, where one unit is defined as the amount of enzymerequired to hydrolyze 1 μmol Cbz-Gly-Pro-pNA (for PEP) orCbz-Gly-Gln-pNA (for a glutamine-specific protease) per min underspecified conditions. Most PEPs have specific activities in the range of5-50 units/mg protein. It will be understood by those of skill in theart that the dose can be raised, but that additional benefits may not beobtained by exceeding the useful dosage. Dosages will be appropriatelyadjusted for pediatric formulation. In children the effective dose maybe lower, for example at least about 0.1 mg, or 0.5 mg. In combinationtherapy a comparable dose of the two enzymes may be given; however, theratio will be influenced by the relative stability of the two enzymestoward gastric and duodenal inactivation.

Enzyme treatment of Celiac Sprue is expected to be most efficacious whenadministered before or with meals. However, since food can reside in thestomach for 0.5-2 h, and the primary site of action is expected to be inthe small intestine, the enzyme could also be administered within 1 hourafter a meal.

Optimal gluten detoxification in vivo can also be achieved by combiningan appropriate gastric protease with a PEP that acts upon glutenpeptides in the duodenum, in concert with pancreatic enzymes. This canbe achieved by co-administration of two enzyme doses, e.g. twocapsules/tablets; via co-formulation of the two enzymes in appropriatequantities; etc. Lyophilized duodenal PEP particles or granules can beprotected by a suitable polymeric enteric coating that promotes enzymerelease only in the duodenum. In contrast, release of the gastricprotease will be initiated immediately upon consumption of the dosageform. Combination treatment involving a PEP and a complementarytherapeutic agent, such as an inhibitor of the enzyme tissuetransglutaminase, is also provided.

In some embodiments of the invention, formulations comprise a cocktailof selected proteases. Such combinations may achieve a greatertherapeutic efficacy. In one combination formulation, Flavobacterium PEPand Myxococcus PEP are co-formulated or co-administered, to allow forthe destruction of a broader range of gluten antigenic peptides.Similarly, combination therapy with one or two PEPs from the above listwith an acid-stable PEP or glutamine endoprotease can lead to moreefficient gluten proteolysis in the stomach, thereby simplifying thetask of gluten assimilation in the upper small intestine.

In another embodiment, the formulation or administration protocolcombines a protease product and an inhibitor of transglutaminase 2(TG2). Such formulations may have additional protection from glutenmediated enteropathy, as TG2 has been shown to have a significantpro-inflammatory effect on gluten peptides in the celiac gut. Inparticular, TG2 inhibitors containing halo-dihydroisoxazole,diazomethylketone or dioxoindole moieties are useful for this purpose.

In another embodiment, the protease or protease cocktail is administeredand/or formulated with an anti-inflammatory agent, e.g. a statin; p38MAP kinase inhibitor; anti-TNFα agent; etc.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific enzyme, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the glutenasesare more potent than others. Preferred dosages for a given enzyme arereadily determinable by those of skill in the art by a variety of means.A preferred means is to measure the physiological potency of a givencompound.

Other formulations of interest include formulations of DNA encodingglutenases of interest, so as to target intestinal cells for geneticmodification. For example, see U.S. Pat. No. 6,258,789, hereinincorporated by reference, which discloses the genetic alteration ofintestinal epithelial cells.

THERAPEUTIC METHODS

The methods of the invention are used to treat foods to be consumed orthat are consumed by individuals suffering from Celiac Sprue and/ordermatitis herpetiformis by delivering an effective dose of glutenase.If the glutenase is administered directly to a human, then the activeagent(s) are contained in a pharmaceutical formulation. Alternatively,the desired effects can be obtained by incorporating glutenase into foodproducts or by administering live organisms that express glutenase, andthe like. Diagnosis of suitable patients may utilize a variety ofcriteria known to those of skill in the art. A quantitative increase inantibodies specific for gliadin, and/or tissue transglutaminase isindicative of the disease. Family histories and the presence of the HLAalleles HLA-DQ2 [DQ(a1*0501, b1*02)] and/or DQ8 [DQ(a1*0301, b1*0302)]are indicative of a susceptibility to the disease.

The therapeutic effect can be measured in terms of clinical outcome orcan be determined by immunological or biochemical tests. Suppression ofthe deleterious T-cell activity can be measured by enumeration ofreactive Th1 cells, by quantitating the release of cytokines at thesites of lesions, or using other assays for the presence of autoimmune Tcells known in the art. Alternatively, one can look for a reduction insymptoms of a disease.

Various methods for administration may be employed, preferably usingoral administration, for example with meals. The dosage of thetherapeutic formulation will vary widely, depending upon the nature ofthe disease, the frequency of administration, the manner ofadministration, the clearance of the agent from the host, and the like.The initial dose can be larger, followed by smaller maintenance doses.The dose can be administered as infrequently as weekly or biweekly, ormore often fractionated into smaller doses and administered daily, withmeals, semi-weekly, or otherwise as needed to maintain an effectivedosage level.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of the invention or to represent that the experiments below areall or the only experiments performed. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, andthe like), but some experimental errors and deviations may be present.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Comparison of PEP Activities

To gain insight into the similarities and differences between naturallyoccurring prolyl endopeptidases, we have systematically compared theproperties of three homologous PEPs from different bacterial sources.Our studies have utilized two known recombinant PEPs from Flavobacteriummeningosepticum (FM) and Sphingomonas capsulata (SC), respectively, anda novel PEP from Myxococcus xanthus (MX) that we have expressed for thefirst time as a heterologous recombinant protein. The enzymaticactivities of these PEPs were quantitatively analyzed versus modelsubstrates as well as two gluten-derived peptides with potentialrelevance to Celiac Sprue pathogenesis. In particular, we have probedthe influence of substrate chain length, pH, pancreatic proteases andintestinal brush border peptidases on the activity of each PEP. Both invivo and ex vivo experiments were performed as part of these studies.

Experimental Procedures

Cloning of PEP Genes. The PEP genes were amplified from the genomic DNAfrom the corresponding bacterial strains (F. meningosepticum: ATCC13253; S. capsulata: ATCC 14666; M. xanthus: ATCC 25232). The sequenceof the putative MX PEP is available from the NCBI database (Locus IDAAD31004). Oligonucleotides used for PCR amplification included: (SEQ IDNO:1) (1) FM first half: 5′-AAC CAA TCA TAT GAA GTA CAA CAA ACT TTC TGTG (NdeI), (SEQ ID NO:2) 5′-GAT AAA AAC GGA AAG CTT GTA AGG GC (HindIII);FM second half: (SEQ ID NO:3) 5′-GCC CTT ACA AGC TTT CCG TTT TTA TC(HindIII) and (SEQ ID NO:4) 5′-CCC TTA ATT TTC AAA TTT TAG CTC GAG TTTATG ATT TAT A (SacI); (2) SC first half: (SEQ ID NO:5) 5′-AGG ATA TCCATA TGA AGA ACC GCT TGT GG (NdeI), (SEQ ID NO:6) 5′-GAC AAC CTC GAA TCCGTC GGC ATT G (HinfI); SC second half: (SEQ ID NO:7) 5′-CAA TGC CGA CGGATT CGA GGT TGT C (HinfI), (SEQ ID NO:8) 5′-CGC GGG GAC CTC GAG TAG AAACTG (SacI); (3) MX: (SEQ ID NO:9) 5′-CT CCC CAT ATG TCC TAC CCG GCG ACC(NdeI) and (SEQ ID NO:10) 5′-GTG GCG GCG CAG GGC CGC AAG CTT CCC AAG CG(HindIII). The amplified genes were cloned into a pET28b plasmid(Novagen).

Expression and Purification of PEPs. Expression plasmids were introducedvia transformation into BL21(DE3) cells. Transformants grown at 37° C.,and induced in the presence of 100 μM IPTG at 22° C. overnight. Lowtemperature induction was found to improve the yield of active enzyme.All purification steps were performed at 4° C. unless noted otherwise.Since FM and SC PEP enzymes naturally possess a signal sequence, theyare secreted into the periplasmic space of E. coli. A modified osmoticshock protocol (EMD Biosciences, CA) was therefore used to obtain anenriched protein lysate containing either PEP. Cell pellets (4 L ofculture) were resuspended in 30 ml of 30 mM Tris-HCl, pH 8, 20% sucroseand 1 mM EDTA, and stirred slowly at room temperature for 10 min. Thesuspension was centrifuged at 10,000 g for 15 min, and the cell pelletwas resuspended in ice-cold dH₂O and stirred slowly on ice for 10 min.The shocked cells were then centrifuged again at 40,000-50000 g for 30min. The supernatant containing the periplasmic proteins was treated for1-2 h with 1 M NaCl solution (to a final concentration of 300 mM NaCl),1 M imidazole solution (to a final concentration 5 mM imidazole) and 1ml of Ni—NTA resin (Qiagen, Calif.). The crude protein was then loadedonto a column containing additional 1 ml of Ni—NTA resin. After thoroughwash steps using the wash buffer (50 mM phosphate, 300 mM NaCl, pH 7.0)with 0-10 mM imidazole, the PEP was eluted with 150 mM imidazole, 50 mMphosphate, 300 mM NaCl, pH 8. FM PEP was further purified on a FPLCsystem (Amersham Pharmacia, NJ) through a HiTrap-SP cation exchangecolumn. Prior to application on the HiTrap-SP column, the protein wasexchanged into 20 mM phosphate buffer (pH 7). Following injection, PEPwas eluted with a salt gradient from 20 mM phosphate, pH 7 (buffer A) to20 mM phosphate, 500 mM NaCl, pH 7 (buffer B) at a flow rate of 1ml/min. MX PEP, a cytosolic protein, was initially purified from awhole-cell lysate via Ni—NTA affinity chromatography (as detailedabove). The protein was further purified on a Superdex 200 gelfiltration column (Amersham) with an isocratic gradient of 20 mM HEPES,2 mM DTT, pH 7.0 at 1 ml/min.

Activity Assays. Post-proline cleavage activity was measured usingZ-Gly-Pro-p-nitroanilide and Succinyl-Ala-Pro-p-nitroanilide (Bachem,Calif.). Z-Gly-Pro-pNA was dissolved in a PBS:water:dioxane (8:1.2:0.8)assay mixture. The concentration of Z-Gly-Pro-pNA was varied from100-600 μM. Although the substrate Z-Gly-Pro-pNA was effective indetecting enzyme activity, its insolubility at higher concentrationsprecluded kinetic measurements under substrate-saturated conditions. Incontrast, Succinyl-Ala-Pro-pNA, had the advantage of high watersolubility at all pH values tested, and was therefore a preferredsubstrate for kinetic studies. Hydrolysis of Suc-Ala-Pro-pNA by FM, SCand MX PEPs was monitored in a reaction mixture (300 μl) consisting of30 μl of 10× PBS buffer, a final concentration of 0.01-0.02 μM enzyme,and Suc-Ala-Pro-pNA (5 mM stock) at final concentrations ranging between100 μM to 4 mM. The release of the p-nitroanilide wasspectrophotometrically detected at a wavelength of 410 nm. The initialvelocity of the reaction was determined by the increase in absorbance at410 nm, which was used to calculate Km and Kcat according to theMichaelis-Menten relationship. For measurement of the influence of pH onthe enzyme activity, a series of pH buffer solutions were prepared usingcitric acid and disodium phosphate for pH values from 3.0 to 6.0, andsodium phosphates for pH values from 7.0 to 8.0. Reaction mixtures (300μl) consisted of 30 μl of 10× pH buffer, final concentration of 0.01 μMenzyme, and Suc-Ala-Pro-pNA to final concentrations between 100 μM to 4mM.

pH Stability. The ability to retain enzyme activity after exposure toacidic environments was determined. Hydrochloric acid solutions (10 μl)at pH values ranging from 1.5 to 4.0 were mixed with 1 μl of enzyme for10-20 min. The acidic mixtures were then neutralized with 40 μl of 10×PBS solution, 60 μl of 5 mM substrate to a final volume of 300 μl. Therecovered enzyme activity was measured spectrophotometrically andcompared with non-acid treated controls under identical conditions.

Gastric and Pancreatic Protease Stability. In a 96-well U-bottomedplate, 5 μL of 2× reaction buffer (40 mM Na₂HPO₄, pH=6.5 for pancreaticenzymes or 20 mM HCl for pepsin) was placed, and 1 μL of the degradingenzyme (either 1 mg/ml pepsin or a cocktail of 1 mg/ml trypsin, 1 mg/mlchymotrypsin, 0.2 mg/ml elastase and 0.2 mg/ml carboxypeptidase A)followed by 4 μL of PEP (5-10 U/ml) were added. The plate was incubatedat 37° C. for various times (e.g. 0, 5, 10, 20 and 30 min), with 190 μLof PEP substrate solution (2 μl Z-Gly-Pro-p-nitroanilide (16.8 mg/ml indioxane) 14 μl dioxane, 24 μl water, 150 μl 10 mM PBS buffer, pH=7.5)added to each well. Absorption was measured at 410 nm for 1 to 2 minevery 10 s to assay residual activity. Each buffer also contained 5mg/ml gluten. Untreated gluten was used for pepsin, whereas glutenpreviously proteolyzed with pepsin (0.01 M HCl, pH=2.0, 1:50 w/w, 2 h,37° C.) was used for all other enzymes. Wells containing acid (pH=2.0)were neutralized by addition of 10 μL 0.1 M NaOH before addition of thePEP substrate. Enzyme activities are expressed as a percentage of themaximum activity, typically observed at the zero time point.

Substrate Specificity. In addition to the reference substrates above,enzyme specificity was also evaluated using two immunogenic peptidesderived from the sequence of γ-gliadin proteins in gluten. Both peptideswere synthesized using solid-phase peptide synthesis. The peptide (SEQID NO:11) PQPQLPYPQPQLP contains the immunodominant γ II-epitope, and isresistant to proteolysis by pepsin or any pancreatic enzyme. PEPspecificity toward this substrate was assessed in a competitive assay inwhich 100 μM (SEQ ID NO:12) PQPQLPYPQPQLP and 100 μM Suc-Ala-Pro-pNAwere mixed and reacted with 0.02 μM PEP at 25° C. The initial velocityof Suc-Ala-Pro-pNA cleavage was measured spectrophotometrically, whereasthe initial velocity of (SEQ ID NO:13) PQPQLPYPQPQLP hydrolysis wasdetermined via HPLC. The apparent specificity, kcat/KM, for thehydrolysis of (SEQ ID NO:14) PQPQLPYPQPQLP could be determined based onthe known kcat/KM of the enzyme for Suc-Ala-Pro-pNA and the observedreaction rates of the two substrates. In addition to PQPQLPYPQPQLP, PEPspecificity for the more complex but physiologically relevant peptide(SEQ ID NO:15) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (33-mer) was alsoassessed. Proteolysis reactions were performed at 37° C. in PBS bufferwith 5-100 μM peptide and 0.1 μM PEP for time periods of 1 min-4 hrs.

The decrease in substrate concentration as well as concomitantintermediate and product build-up were monitored with HPLC analysis.RP-HPLC was performed on a system consisting of Beckman or RaininDynamax SD-200, a Varian 340 UV detector set at 215 nm and 280 nm.Solvent A was H2O with 0.1% TFA and solvent B was acetonitrile with 0.1%TFA; gradient used: 0-5% B in 0-15 min, 5-30% B in 15-30′, 30-100% B in30-35 min, 100% B for 5′; flow 1 ml/min; separation was performed on a4.6×150 mm reverse phase C-18 column (Vydac, Hesperia, Calif., USA).Samples were centrifuged for 10 min at 13,400 g, prior to injection of10-100 μl. Both (SEQ ID NO:11) PQPQLPYPQPQLP as well as the 33-mer havemultiple post-proline endoproteolytic sites. Thus, multiple peptidesaccumulate during the course of the reaction, some of which aresecondary PEP substrates in themselves. Electrospray-Ion Trap-MS-MScoupled with a UV-HPLC (LCQ Classic/Surveyor, ThermoFinnigan, CA) wasused to identify the preferred cleavage sites in (SEQ ID NO:11)PQPQLPYPQPQLP and the 33-mer.

For further evaluation of the proteolysis of the 33-mer and (SEQ IDNO:11) PQPQLPYPQPQLP in the appropriate physiological environment,gluten (30g/L) was suspended in 0.01 M HCl (pH=2.0) and incubated in thepresence of pepsin (600 mg/L) for 2 h at 37° C. The resulting solutionwas neutralized using 10 M NaOH and diluted to 10 g/L in a phosphatebuffer (40 mM, pH 6.5). 25 μl of this suspension were then supplementedwith the 33-mer (0.1 mg/ml), (SEQ ID NO:11) PQPQLPYPQPQLP (0.08 mM),trypsin (0.1 mg/ml), chymotrypsin (0.1 mg/ml), elastase (0.02 mg/ml),carboxypeptidase A (0.02 mg/ml). Prolyl endopeptidase (FM or MX; 1×: 500mU/ml; 5×: 2.5 U/ml; 10×: 5 U/ml) and rat intestinal brush bordersurface membranes (BB, 1×: 40 mU/ml, 2×: 80 mU/mi, DPP IV activity) wereadded to a total volume of 150 μl. The mixture was incubated at 37° C.and 25 μl aliquots were taken at 0, 5, 10, 30 and 60 min and immediatelyheat deactivated.

To examine the chain length specificity of individual PEPs, we performedcompetitive reactions containing both gluten-derived peptides, subjectedthe reaction mixture to RP-HPLC, and monitored the disappearance of eachsubstrate was monitored as a function of time. The peak areas of the33-mer (32.5 min) and (SEQ ID NO:11) PQPQLPYPQPQLP (27.5 min) wereintegrated.

In Vivo Endopeptidase Activity. An adult (female or male) rat wasanesthetized and maintained at 36-37° C. during the entire surgicalprocedure. The peritoneal cavity was opened, and a small incision wasmade at the beginning and the end of a 15-20 cm jejunum segment.Polyethylene catheters were inserted and secured into the two ends. Theinput catheter was connected with a pump-driven syringe filled with asolution. The jejunum segment was perfused initially with PBS buffer toremove any residual debris at a flow rate of 0.4 ml/min. Purifiedpeptide solutions (peptide concentration ranges from 25-100 μM) werethen perfused at 0.4 ml/min through the jejunum segment with a 10-40 minresidence time. In the case of a co-perfusion, the input catheter isconnected with two simultaneous syringes, one with a peptide solutionand the other with the prolyl endopeptidase solution (concentrationranges from 50-500 μU/μl). Fluid from the output catheter was collectedinto small centrifuge tubes in dry ice for subsequent analysis. Thecollected digestive products were analyzed by HPLC on a C18 column.

Results

PEP Protein Expression. FM and SC PEPs have their own signal sequences,and were therefore expressed as secreted, soluble enzymes in theperiplasmic space of E. coli. A simple freeze-thaw lysis procedure ledto recovery of periplasmic protein without significant contamination bycytoplasmic proteins. In contrast, the MX PEP lacks a native signalsequence, and was therefore expressed as a cytoplasmic protein. PEP waspurified from each lysate by Ni—NTA affinity purification, followed by asecond chromatographic step. The yields of active FM, SC and MX PEPswere 1 mg/L, 60 mg/L and 30 mg/L, respectively. The purity of thevarious PEPs was determined by SDS-PAGE to be >90%.

Kinetic Analysis with Reference Substrates. The activity of each PEP wasinitially evaluated using the standard chromogenic substratesuccinyl-Ala-Pro-pNA. Release of the p-nitroaniline was detected at 410nm, and kinetic data was fitted to the Michaelis-Menten relationship.Succinyl-Ala-Pro-pNA was selected as a reference substrate instead ofthe more commonly used Z-Gly-Pro-pNA due to the low solubility of thelatter substrate, which necessitated use of co-solvents. The calculatedkcat and KM values of FM, MX and SC PEPs for succinyl-Ala-Pro-pNA aretabulated (Table 1). While these enzymes all exhibited comparable levelactivity to that of a serine protease, MX PEP has a higher specificitythan the FM PEP, whereas SC PEP has an intermediate level of specificity(Table 2). The higher specificity of MX can be attributed mainly to itshigher affinity for the substrate, as reflected in the K_(M).

TABLE 1 Kinetic parameters for Succinyl-Ala-Pro-p-nitroanilidehydrolysis by FM PEP, MX PEP and SC PEP. K_(cat) (s⁻¹) K_(M) (mM)K_(cat)/K_(M) (mM⁻¹/s⁻¹) FM PEP 33 0.91 37 MX PEP 51 0.35 146 SC PEP 1442.1 67

TABLE 2 Specificity of FM PEP, MX PEP and SC PEP for the immunogenicgliadin peptide (SEQ ID NO: 4) PQPQLPYPQPQLP. K_(cat)/K_(M) (mM⁻¹/s⁻¹)FM PEP 178 MX PEP 548 SC PEP 492

Enzyme Activity vs. pH. The luminal environment of the duodenum isapproximately at pH 6. Therefore, a therapeutically useful PEP mustretain high specific activity at that pH. The steady state turnoverrate, kcat, of each PEP was titrated in various pH conditions using100-4000 μM succinyl-Ala-Pro-pNA, shown in FIG. 1. Both FM PEP and MXPEP exhibited active site pKa around pH 6, indicating optimal activityin the pH 6-8 range. The diminished activity of both enzymes at pH 5 isconsistent with the well-established role of a histidine residue as thegeneral base in the serine protease catalytic triad, but alternativelyit may indicate a change from the active enzyme conformation to aninactive state. Such conformational changes have been implicated in thecatalytic cycle of the structurally characterized porcine brain PEP.Interestingly, the SC PEP, which has the broadest pH profile, shows amarked increase in maximum velocity under weakly basic conditions.

PEP Stability. Although orally administered therapeutic proteins can beformulated to protect them from the acidic and proteolytic environmentof the stomach, intrinsic acid stability of a PEP is likely to be adesirable characteristic in its use as a therapeutic agent for CeliacSprue. We therefore evaluated the extent to which the activity of eachPEP remains intact after 10 min of incubation at selected pH valuesbetween 1.6 and 3.9. Within this pH range, the FM PEP retained 50-70% ofits original activity; the MX PEP retained 70-90% activity; and the SCPEP retained 30-80% activity. Thus, although all PEPs appear to bemoderately acid-stable, the MX PEP is most versatile. Since therapeuticefficacy would require a PEP to act upon gluten in conjunction withpancreatic proteases that are secreted into the duodenum, the resistanceof FM PEP and MX PEP toward both gastric and pancreatic enzymes wasevaluated. For this we pre-incubated the enzymes with physiologicalquantities of either pepsin (at pH 2) or a cocktail comprising oftrypsin, chymotrypsin, elastase and carboxypeptidase A (at pH 6.5). Ascan be seen in FIG. 2, both FM and MX PEP were highly susceptible topepsin catalyzed proteolysis, whereas they appear to be remarkablystable to destruction in the presence of physiological quantities of thepancreatic enzymes.

Kinetic analysis using PQPQLPYPQPQLP as a substrate. The immunogenicpeptide PQPQLPYPQPQLP is a recurring sequence in γ-gliadins, and isresistant to proteolysis by gastric and pancreatic proteases. It is alsohighly resistant to digestion by intestinal brush border peptidases,with only dipeptidyl carboxypeptidase I (DCP1) able to act upon it.Treatment of this peptide with PEP results in cleavage at internalproline residues, which in turn generates new recognition sites forbrush border aminopeptidases. Thus, (SEQ ID NO:11) PQPQLPYPQPQLPrepresents a good test substrate to probe PEP specificity.

The k_(cat)/K_(M) values of each PEP were determined in an assay mixturecontaining (SEQ ID NO:11) PQPQLPYPQPQLP as well as Suc-Ala-Pro-pNA as acompeting substrate. The rates of disappearance of both substrates weredetermined by independent detection methods. The initial rate ofdisappearance of (SEQ ID NO:11) PQPQLPYPQPQLP was measured by HPLC,whereas the rate of consumption of Suc-Ala-Pro-pNA was measuredspectrophotometrically. Both FM and MX PEP had a 5-fold higherspecificity for the gluten peptide as compared to the chromogenicsubstrate, whereas the SC PEP showed a 7-fold increase in specificityfor the gluten peptide (Table 2). This increase in specificity suggeststhat longer peptides may provide additional anchors at the catalyticsite, a hypothesis that is consistent with the observation thatAla-Pro-pNA (which lacks an N-terminal succinyl group or a carboxybenzylgroup) did not react with any of the PEPs.

To analyze the regiospecificity of hydrolysis of (SEQ ID NO:11)PQPQLPYPQPQLP by individual PEPs, samples corresponding to early timepoints were further analyzed by LC/MS/MS. The results, shown in FIG.3A-3D, reveal that each PEP has unique subsite preferences. While thepreferred site of cleavage by FM PEP was at the (SEQ ID NO:11)PQPQLPPYP|QPQLP position, MX PEP preferentially cleaved the same peptideat the (SEQ ID NO:11) PQPQLP|YPQPQLP position. SC had comparablepreference for either site of cleavage. All enzymes preferentiallycleaved the peptide at a proline located near the middle of thesequence, highlighting their functional difference from prolyl-specificexopeptidases such as DPP IV.

Chain Length Tolerance and Selectivity. It has been suggested thatprolyl endopeptidases from the serine protease family are limited withregard to chain lengths of potential substrates. To test this hypothesisin the context of the three bacterial PEPs studied here, we comparedtheir hydrolytic activities against a physiologically relevant 33-merpeptide sequence from wheat gliadin, LQLQPFPQPQLPYPQPQLPYPQPQLP YPQPQPF(FIG. 4A). The FM PEP (0.1 μM) was able to hydrolyze 10 μM of the 33-merin about 2-3 minutes, whereas the SC PEP required >1 hr to reach acomparable endpoint. Based on initial rates, the FM PEP was estimated toact 5-fold faster on the 33-mer than the MX PEP, and >20 fold fasterthan the SC PEP. Thus, the SC PEP appears to have a severe chain lengthrestriction for long peptide substrates.

The intermediates and products from hydrolysis of the 33-mer by the FMand MX PEPs were analyzed by LC/MS/MS (FIG. 4B-C). Several features arenoteworthy. First, even at relatively early time-points, the digestiveproducts of the MX PEP were predominantly small fragments, whereas FMPEP digestion yielded a significant pool of long intermediates such asLQLQPFPQPQLPYPQPQLP, LQLQPFPQPQLPYP and LQLQPFPQPQLP. Thus, althoughboth PEPs are able to effectively proteolyze the 33-mer, they havedistinct hydrolytic patterns on this complex substrate. In particular,either the MX PEP appears to be processive (i.e. for each 33-mersubstrate molecule, it sequentially cleaves all the preferred sites inthe chain prior to release), or alternatively the enzyme has a strongbias toward shorter chain substrates. It could also be noted that theC-terminal fragments generated by the two enzymes are different (QPQPFfor the FM PEP, and YPQPQPF for the MX PEP). This finding is consistentwith observed sub-site preference in the case of (SEQ ID NO:11)PQPQLPYPQPQLP digestion.

To directly investigate chain length selectivity of the three enzymes,we co-incubated (SEQ ID NO:11) PQPQLPYPQPQLP andLQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF with each PEP (FIG. 6A-C) Both the SCPEP and the MX PEP showed a clear preference for the 13-mer peptide,whereas the FM PEP showed comparable selectivity for both peptides.

To further evaluate the substrate preferences, (SEQ ID NO:11)PQPQPLPYPQPQLP and the 33-mer were mixed with pepsin-treated gluten, andallowed to react with pancreatic enzymes in the presence of BBM andeither FM PEP or MX PEP. As seen in the HPLC traces (FIG. 6A-B), the33-mer had the longest retention time, whereas (SEQ ID NO:11)PQPQLPYPQPQLP and other medium-length gluten peptides eluted earlier.Here too the FM PEP proteolyzed (SEQ ID NO:11) PQPQLPYPQPQLP, the 33-merand other gluten peptides at comparable rates (FIG. 6A). In the MX PEPdigestion, PQPQLPYPQPQLP and other smaller peptides were rapidly brokendown (in 10 minutes), whereas hydrolysis of the 33-mer occurred at aslower rate (FIG. 6B).

In Vivo Hydrolysis. To validate the implications of the abovebiochemical observations for peptide digestion in the intact smallintestine, each PEP was co-perfused in the rat jejunum with the 33-merpeptide substrate, and the effluent collected at a distance of 15-20 cmfrom the point of perfusion was analyzed. In this live animal model, theimpact of concerted action of the perfused (luminal) PEP and the brushborder (surface) peptidases is assessed. As shown by the in vitroresults above, while the BBM enzymes were insufficient to process the33-mer, FM PEP promoted more complete breakdown of the 33-mer than boththe MX and the SC PEP (FIG. 7). Within a PEP dose range of 50-500 μU/μl,the extent of 33-mer hydrolysis increased with increasing PEP dose,demonstrating that higher doses of PEP could accelerate gluten breakdownin the mammalian gut.

In light of recent findings that related the strong antigenicity ofgliadin peptides to their exceptional digestive resistance, prolylendopeptidases were identified as a potentially interesting family ofenzymes for oral Celiac Sprue therapy. Understanding the enzymologicalproperties of these enzymes is an essential prerequisite for such use.In the above study, prolyl endopeptidases from three bacterial sourceswere selected and expressed in E. coli as recombinant proteins, and weresubsequently purified and characterized. Two of these enzymes (from F.meningosepticum and S. capsulata) have been reported earlier, whereasthe third enzyme (from M. xanthus) represents a new member of the prolylendopeptidase family.

In order to examine the endoproteolytic properties of these enzymes, itis important to utilize peptide substrates with internal cleavage sites.Although model substrates such as Z-Gly-Pro-pNA or Suc-Ala-Pro-pNA havebeen frequently used to identify and characterize polyl endopeptidases,these substrates alone do not provide adequate insight to differentiateendopeptidases from each other or from proline-specific aminopeptidases(such as dipeptidyl peptidase IV (DPP IV)). In the context of CeliacSprue, two peptides (PQPQLPYPQPQLP andLQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) have been recognized as useful probesfor studying the fundamental properties of prolyl endopeptidases, aswell as for their potential for detoxifying gluten. The peptidePQPQLPYPQPQLP contains an epitope found in γ-gliadins that has beenshown to play an immunodominant role in the T cell mediated response togluten in the Celiac gut. It cannot be cleaved by any gastric orpancreatic proteases and is also highly resistant to digestion byintestinal brush border membrane (BBM) peptidases, with only dipeptidylcarboxypeptidase I able to act upon it at a very limited rate. Thus, theefficiency of intestinal metabolism of this peptide can be expected toimprove in the presence of an exogenous prolyl endopeptidase, as hasbeen verified in this study. Treatment of this peptide with PEP resultsin cleavage at an internal proline residue, which in turn generates anew recognition site for brush border aminopeptidases. Thus,PQPQLPYPQPQLP represents a good probe for PEP specificity.

The 33-mer gliadin peptide LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF wasselected as a complementary probe for these studies, because it is astable, physiologically derived product of gastric and pancreaticdigestion of γ-gliadin, and strongly stimulates proliferation ofgluten-reactive T cells from virtually all Celiac Sprue patients testedthus far. Therefore, endoproteolytic breakdown of this 33-mer peptiderepresents an especially challenging goal for an exogenous PEP. Likemost other antigenic gluten peptides, the 33-mer contains multipleproline residues, and can be expected to present more than one cleavagesite to a PEP. At the same time its multivalent character suggests thatPEP action alone is unlikely to eliminate all residual antigenicity ofthis peptide. Consequently, combined action of a PEP and the endogenouspeptidases of the intestinal brush border membrane is required forimmunological neutralization and dietary assimilation of this longproline-rich peptide.

Our investigations into the molecular recognition features of threebacterial PEPs for two gliadin peptides have revealed at least twointeresting and potentially important characteristics of these enzymes.First, although all three PEPs tested here exhibited high specificactivity against reference chromogenic substrates (Table 1), they showedremarkable differences in chain length specificity (FIG. 3A-C). WhereasSC PEP and MX PEP had higher specificity for PQPQLPYPQPQLP than FM PEP(Table 2), the reverse was true for the longer 33-mer gliadin peptide(FIG. 4A), especially in the case of the SC PEP, which had extremelypoor activity against the 33-mer.

Structural and biochemical analysis led to the proposal that theactivity of PEPs is limited to substrates containing fewer than 30 aminoacid residues. In that light the good activity of MX PEP and especiallyFM PEP against the 33-mer peptide is surprising. The broad chain lengthtolerance of FM PEP is vividly demonstrated in competitive in vitro andin vivo assays, where FM PEP was able to process longer and shortersubstrates at comparable rates. Second, sequence analysis of the majorproteolytic products derived from both gliadin substrates demonstratedthat the PEP's had distinct sub-site specificity as well asregiospecificity in the context of the longer repetitive sequence. Forexample, the FM PEP preferentially cleaved at PQPQLPYP|QPQLP, whereasthe MX PEP preferred the PQPQLP|YPQPQLP site, and the SC PEP hadcomparable activity toward either site.

Similarly, sequence analysis of initial hydrolytic products of the33-mer peptide underscored regiochemical differences between FM PEP andMX PEP. Whereas MX PEP treatment generated fragments mostly of 4-5residues (presumably processed sequentially from both termini), FM PEPyielded longer intermediates (presumably as a result of a preferentialcleavage near the center of the peptide). Thus, the active sites ofthese enzymes are clearly different, which in turn has potentialimplications for the use of these enzymes detoxifying dietary gluten fora Celiac Sprue patient.

In addition to analyzing substrate specificity, we have alsoinvestigated other therapeutically relevant properties of our set ofthree PEPs. They include pH dependence of enzyme activity, acidtolerance of the protein, and resistance toward inactivation by gastric,pancreatic and intestinal proteases/peptidases. All enzymes have a pHactivity profile that is well matched to the mildly acidic environmentof the upper small intestine (pH 6-6.5). They also appear to bemoderately stable toward acid exposure as well as pancreatic protease(but not pepsin) action, with the MX PEP being the most stable. Theenzymes also retain activity in the intact small intestinal lumen of arat, indicative of their stability toward both intestinal secretions aswell as brush border membrane peptidases. Finally, the expression levelsof these enzymes vary significantly in recombinant E. coli.Specifically, in comparison to the FM PEP, the expression levels of SCand MX PEPs were substantially superior.

The porcine brain PEP has a didomain architecture, including an unusualβ-propeller domain that appears to regulate proteolysis. Pairwisesequence alignments between this structurally characterized PEP and FM,MX and SC PEP reveal 39% (49%), 36% (45%) and 40% (48%) identity(similarity), respectively. These alignments also suggest that thebacterial PEPs are comprised of a catalytic and a β-propeller domain.Since their active sites are predicted to lie near the interface betweenthe two domains, mutagenesis at the inter-domain interface could alterprotein dynamics and in turn affect substrate tolerance and specificity.

The above results provide a basis for protein engineering efforts of PEPenzymes. This family of serine proteases includes numerous otherputative homologs whose cDNAs have been sequenced but whose geneproducts remain to be characterized. In light of the favorableproperties of the MX PEP, which was expressed and characterized for thefirst time as part of this study, it will be useful to screen additionalwild-type enzymes.

Example 2 Heterologous Expression of PEP in Lactobacilli

In one embodiment of the present invention, a Celiac Sprue patient isprovided with a recombinant organism modified to express a PEP of theinvention. The recombinant organism is selected from those organismsthat can colonize the intestinal mucosa without detriment to thepatient, thereby providing an endogenous source of PEP to the patient.As one example, Lactobacilli such as L. casei and L. plantarium cancolonize the intestinal mucosa and secrete PEP enzymes locally. Giventheir widespread use in food processing, they can also be used as anefficient source of PEP for industrial (to treat foodstuffs) and medical(to prepare PEP for pharmaceutical formulation) use. PEPs are expressedin such lactobacilli using standard recombinant DNA technologies. Forexample, Shaw et al. (Shaw, D M, Gaerthe, B; Leer, R J, Van der Stap, JG M M, Smittenaar, C.; Den Bak-Glashouwer, Heijne, M J, Thole, J E R,Tielen F J, Pouwels, P H, Havenith, C E G (2000) Immunology 100,510-518) have engineered Lactobacilli species to express intracellularand surface-bound tetanus toxin. The intact PEP genes (including leadersequences for efficient bacterial secretion) are cloned into shuttleexpression vectors such as pLP401 or pLP503 under control of the(regulatable) amylase promoter or (constitutive) lactate dehydrogenasepromoter, respectively. Alternatively, recombinant food gradeLactobacilli strains are generated by site specific recombinationtechnology (e.g. see. Martin M C, Alonso, J C, Suarez J E, and Alvarez MA Appl. Env. Microbiol. 66, 2599-2604, 2000). Standard cultivationconditions are used for Lactobacilli fermentation, such as thosedescribed by Martin et al.

Example 3 Heterologous Expression of PEP in Yeasts

Both naturally occurring and recombinant cells and organisms are used toproduce the glutenases useful in practice of the present invention.Preferred glutenases and producing cells include those from organismsknown to be Generally Regarded as Safe, such as Flavobacterium,Aeromonas, Sphingomonas, Lactobacillus, Aspergillus, Xanthomonas,Pyrococcus, Bacillus and Streptomyces. Extracellular glutenase enzymesmay be obtained from microorganisms such as Aspergillus oryzae andLactobacillus casei. Preferred cells include those that are already usedin the preparation of foodstuffs but have been modified to express aglutenase useful in the practice of the present invention. As oneexample, yeast strains such as Saccharomyces cerevisiae are useful forhigh level expression of secreted heterologous proteins. Genes encodingany of the PEPs described above (mature protein only) are cloned inexpression plasmids designed for optimal production of secretedproteins. An example of such a heterologous expression strategy isdescribed in Parekh, R. N. and Wittrup, K. D. (Biotechnol. Prog. 13,117-122, 1997). Either self-replicating (e.g. 2 micron) or integrating(e.g. pAUR101) vectors can be used. The GAL1-10 promoter is an exampleof an inducible promoter, whereas the ADH2 promoter is an example of aconstitutive promoter. The cDNA encoding the mature PEP is fuseddownstream of a leader sequence containing a synthetic pre-pro regionthat includes a signal cleavage site and a Kex2p cleavage site. S.cerevisiae BJ5464 can be used as a host for production of the peptidase.Shake-flask fermentation conditions are described by Parekh and Wittrupin the above-cited reference. Alternatively, high cell density fed-batchcultures can be used for large scale production of the peptidases; arepresentative procedure for this purpose is described in Calado, C. R.C, Mannesse, M., Egmond, M., Cabral, J. M. S. and Fonseca, L. P.(Biotechnol. Bioeng. 78, 692-698, 2002).

Example 4 Enteric Capsule Formulation of Prolyl Endopeptidase

Gelatin capsules are filled with 100 mg Myxococcus xanthus prolylendopeptidase and 10 mg of silicon dioxide. The capsules are entericallycoated with Eudragit polymer and put in a vacuum chamber for 72 hours.The capsules are then held at a range of temperature of 10° C. to 37° C.and a controlled humidity level of 35-40%.

Example 5 Studies of Enteric Capsule Formulation of Prolyl Endopeptidase

A study is conducted where patients with Celiac Sprue are enrolled in atwo week-long study. Gelatin capsules containing 90% Myxococcus xanthusprolyl endopeptidase mixed with 10% silicon dioxide are used. Thecapsules are hand-filled with the mixture, banded, and coated with a 10%Sureteric enteric coating (a polymer of polyvinylacetatephthalatedeveloped by the Canadian subsidiary of Merck & Company). Samples areacid-tested by exposing the coating to 1 N HCL for one hour in order tosimulate the acid environment of the stomach. The capsules are then putin a vacuum chamber for 72 hours.

Two 100 mg capsules are administered to each patient prior to each meal.The patients are instructed to eat all kinds of food without abstainingfrom those that were known to cause distress, e.g., bloating, diarrhea,and cramps.

Example 6 Enteric Pill Formulation of Prolyl Endopeptidase

400 mg of L-tartaric acid and 40 mg of polyethylene glycol-hydrogenatedcastor oil (HCO-60) are dissolved in 5 ml of methanol. This solution isplaced in a mortar previously warmed to 30° C. To the solution is added100 mg of Myxococcus xanthus prolyl endopeptidase. Immediately after theaddition of PEP, the mixture is stirred with a pestle under a hot aircurrent (40° C.) and then placed in a desiccator under vacuum overnightto remove the solvent. The resulting solid-mass is pulverized with apestle and kneaded with 30 mg of sodium bicarbonate and a small amountof 70% ethanol. The mixture is then divided and shaped into pills ofabout 2 mm size and thoroughly dried. The dried pills are given acoating of hydroxypropylmethylcellulose phthalate (HP-55) to obtain anenteric formulation.

Example 7 Endoprotease Activity

The gene for an endoprotease (EPB2; PubMed accession number U19384, nt94-1963) from barley (Hordeum vulgare subsp. vulgare) was subcloned intoa pET 28 b (Invitrogen) vector using BamH1 and EcoR1 insertion sites;the resulting plasmid was designated pMTB1. An inactive 43 kDaproprotein form of EPB2 was expressed from pMTB1 in the cytoplasm ofBL21 E. coli cells. The proprotein was solubilized from the inclusionbodies using 7 M urea. The solubilized protein was purified on a Ni—NTAcolumn. Auto-activation of proEPB2 to its mature, active form wasachieved by addition of citrate-phosphate buffer, pH 3 (prepared bymixing 0.1 M sodium citrate and 0.2 M sodium phosphate). Under suchacidic conditions, proEPB2 converts rapidly into a mature form with amolecular weight of 30 kDa (FIG. 2). By 72 hours, mature EPB2 undergoesautolysis. N-terminal sequencing yielded an N-terminal sequencebeginning with VSDLP.

Under acidic conditions, the mature form of EPB2 efficiently digestspurified α2-gliadin, a source of peptides that are immunogenic to peoplewho suffer from Celiac Sprue. The cysteine proteinase inhibitor,leupeptin, inhibits this activity, confirming its mechanism as acysteine protease. The pH optimum of proEPB2 activation and α2-gliadindigestion is 2.4-3.5, which can therefore provide a treatment for CeliacSprue consisting of oral administration of proEPB2.

Example 8 Formulation and Efficacy Analysis of M. xanthus PEP

Lyophilization of M. xanthus PEP was performed as follows. The PEP waspurified as described in Example 1, and concentrated to an initialconcentration of 7.7 mg/ml by Tangential-Flow Filtration (TFF) using a10K MWCO Pellicon difiltration membrane (Millipore, PLCGC10, 50 cm, Cat.No. PXC010C50). TFF (using a LabScale TFF from Millipore, Cat. No.29751) was performed for approximately 12 hours (pressure of 50 psi(retentate)/30 psi (permeant)), with periodic addition to the reservoirof 50 mM Sodium Phosphate, 3% Sucrose pH 7.5. Thereafter, PEG-4000 wasadded with a target concentration of 1%. The final protein concentrationwas 70-100 mg/ml. This material was centrifuged, then lyophilized. Thelyophilization was performed in square petri dishes (Falcon Cat. No.35-1112) in a DuraStop lyophilizer using parameters outlined in theTable below. Typically, 0.7-0.85 mg PEP was present per mg oflyophilized material. No loss of specific activity of the PEP wasobserved upon lyophilization.

Step Temperature Pressure Duration Ramp Rate Freezing 1 −50° C.Atmospheric 2 hrs 0.3° C./minute Annealing −35° C. Atmospheric 3 hrs0.3° C./minute Freezing 2 −50° C. Atmospheric 2 hrs 0.3° C./minute 1°Drying −20° C. 100 mTorr 16.9 hrs 0.5° C./minute 2° Drying +25° C. 100mTorr 8.0 hrs 0.2° C./minute *P. Temp. = Avg. Product Temp. at end ofstep. **1° = Primary Drying. ***2° = Secondary Drying

Blending for the M. xanthus PEP was performed as follows. Lyophilizedcakes were pulverized to a light powder. All samples were weighed forrecovery and stored in sealed 50 mL conical vials at 4° C. A blend wasprepared as shown below. The excipients were selected to provide properflow and disintegration properties for the blended mixture.

Order of Addition Excipient Percentage 1 Lyophilized enzyme 63%cake/powder 2 Calcium Silicate 2% 3 Talc 5% 4 Crospovidone 5% 5 Avicel25%

The lyophilized enzyme and excipients were blended in a V-blender forseveral hours. The material was then used to make enteric-coatedcapsules or tablets. 100-150 mg M. xanthus PEP could be loaded into asingle hard gelatin capsule, size 00 (Capsugel). Alternatively, Vcapvegetable capsules (size 00, Capsugel) can also be used with no impacton enzyme activity.

For enteric coating of the capsules, an enteric coating solution wasprepared as shown below:

Order of Addition Excipient Amount added 1 RODI water 49.5 mL 2 Talc 8.1g 3 Eudragit L50 D-55 111.0 mL 4 Triethyl Citrate 1.62 mL

The enteric coating was mixed vigorously in a beaker on a stir plate.The solution was then decanted into a spray bottle. Rat capsules werecarefully spread on paper towels in groups of 20 and the enteric coatingsolution sprayed onto the capsules. Warm air was used to partially drythe capsules before moving them to a dry paper towel where theyair-dried for 30 minutes before the next coat was applied. A total of 3coats were applied in order to cover all sides of the capsules. Thesewere air dried several hours before being transferred to a storagecontainer. Although some activity of the PEP is lost as a result ofenteric coating, a substantial fraction of the activity is retained, andis stable for at least 1 month at 4 C. storage.

An alternative method to formulate the enzyme for intestinal delivery isas an enteric-coated tablet. Tablets have the advantage of more rapiddissolution in the weakly acidic environment of the upper smallintestine. Another advantage of the tablet formulation is that moreenzyme can be compacted into a smaller volume than for a capsule. Theirprimary liability is that proteins frequently denature under highpressures. In a method of tablet preparation of M. xanthus PEP, the samelyophilized blend as above was used. Tablets were prepared at a punchstrength of 3000 psi held for 15 seconds. No activity was lost in theprocess, demonstrating the feasibility of tablet formulations of thisenzyme.

To test the efficacy of the enteric-coated oral capsule formulationdescribed above, two types of tests were performed. In vitro dissolutiontests were performed on a Hanson SR8-Plus Dissolution Tester usingSimulated Gastric Fluid (SGF; 2 g/L NaCl, pH 1.2, adjusted using 6 NHCl) and Simulated Intestinal Fluid (SIF; 6 g/L monobasic potassiumphosphate with or without 10 g/L pancreatin, pH 6.8, adjusted using 5 NNaOH). Enteric coated capsules were first tested for resistance todissolution in SGF for up to 2 h at 37° C. No protein release was noted.Subsequently the capsules were subjected to similar dissolution tests inSIF at 37° C. A substantial fraction of the encapsulated material wasreleased in 15 min. By 30 min the material had been completely released.

In vivo tests of the capsules were performed in rats using smaller hardgelatin capsules (Size 9 capsules, Torpac). Approximately 16 mg of thelyophilized formulation blend was encapsulated in each enteric-coatedcapsule, corresponding to ˜7 mg PEP. Rats fasted overnight wereadministered via oral gavage one PEP or placebo capsule along with ameasured quantity (300 mg gluten/kg body weight) of gluten syrupprepared as follows. 300 g commercially available wheat gluten flour(Bob's Red Mill, Milwaukie Oreg.) was added to 10 L of a 0.01 M HClsolution to achieve a pH of 2.0. Pepsin (6.0 g, American Laboratories)was added. After incubation at 37° C. for 1 h, the pH was adjusted to2.0 by addition of 35 ml 1 M HCl. After maintenance for an additional 2h at 37° C., the solution was neutralized by addition of 35 g ofNa₂HPO₄, and the pH was adjusted to 7.9 with 10 M NaOH (32.5 ml).Trypsin/Chymotrypsin powder (3.75 g) (Enzyme Development Corp; 1000USP/mg in trypsin, 1000 USP/mg in chymotrypsin) was then added, thereaction maintained at 37° C. for 2 hours, pH 7.9 (pH re-adjustment to7.9 after 1 hour, with 10 M NaOH) and heated at 100° C. for 15 minutesto inactivate the enzymes. The final gluten solution was filteredthrough cheesecloth to remove residual large particles. One PEPcapsule-fed animal and one sham capsule-fed animal was sacrificed after45 min and 90 min each, and the small intestinal contents were analyzedfor gluten content via C18 reversed phase HPLC. Chromatograms werenormalized for total protein content in each sample. Top=45 min,Bottom=90 min (green=placebo, blue=PEP capsule).

Gluten-derived peptides elute in the 20-30 min region. At 45 min as wellas 90 min, the pepsin-trypsin-chymotrypsin treated gluten was minimallymetabolized in the sham-fed animals, whereas it appears to beextensively metabolized. Together, these results indicate thatenteric-coated PEP capsules can survive the gastric environment of thestomach, and catalyze proteolysis of dietary peptides in the smallintestine.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication, patent, or patent application were specifically andindividually indicated to be incorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the inventor to comprise preferredmodes for the practice of the invention. It will be appreciated by thoseof skill in the art that, in light of the present disclosure, numerousmodifications and changes can be made in the particular embodimentsexemplified without departing from the intended scope of the invention.Moreover, due to biological functional equivalency considerations,changes can be made in methods, structures, and compounds withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

1. A formulation for use in treatment of Celiac Sprue and/or dermatitis herpetiformis, comprising: an effective dose of glutenase and a pharmaceutically acceptable excipient.
 2. The formulation according to claim 1, wherein said glutenase is a prolyl endopeptidase.
 3. The formulation according to claim 2, wherein said prolyl endopeptidase is Flavobacterium meningosepticum PEP, Sphingomonas capsulata PEP, or Penicillium citrinum PEP.
 4. The formulation according to claim 2, wherein said prolyl endopeptidase is Myxococcus xanthus PEP.
 5. The formulation according to claim 1, wherein said glutenase is a glutamine specific protease.
 6. The formulation according to claim 5, wherein said glutamine specific protease is Hordeum vulgare endoprotease, Aspergillus oryzae X-Pro dipeptidase, or Aspergillus saitoi carboxypeptidase.
 7. The formulation according to claim 1, wherein said glutenase is stable to acid conditions.
 8. The formulation according to claim 1, wherein said formulation is suitable for oral administration.
 9. The formulation according to claim 1, wherein said formulation comprises an enteric coating.
 10. A composition, comprising: at least about 50% by weight Myxococcus xanthus PEP.
 11. The composition according to claim 10, wherein said Myxococcus xanthus PEP is present at a concentration of at least 1 mg/ml.
 12. The composition according to claim 10, wherein said Myxococcus xanthus PEP is purified by affinity chromatography.
 13. The composition according to claim 10, wherein said Myxococcus xanthus PEP is lyophilized.
 14. The composition according to claim 10, wherein said Myxococcus xanthus PEP is formulated in a pharmacologic unit dose.
 15. A method of treating gluten intolerance, the method comprising: administering to a patient an effective dose of a glutenase; wherein said glutenase attenuates gluten toxicity in said patient.
 16. The method according to claim 15, wherein said glutenase is provided in a formulation according to any one of claims 1 to
 9. 17. The method according to claim 15, wherein said glutenase is admixed with food.
 18. The method according to claim 15, wherein said gluten intolerance is associated with Celiac Sprue or dermatitis herpetiformis. 